Sample records for live microscopy film

This paper reports preliminary results of the observation by acoustic microscopy of living cells in vitro. The scanning acoustic microscope uses high-frequency sound waves to produce images with submicrometer resolution. The contrast observed in acoustic micrographs of living cells depends on the acoustic properties (i.e., density, stiffness, and attenuation) and on the topographic contour of the cell. Variation in distance separating the acoustic lens and the viewed cell also has a profound effect on the image. When the substratum is located at the focal plane, thick regions of the cell show a darkening that can be related to cellular acoustic attenuation (a function of cytoplasmic viscosity). When the top of the cell is placed near the focal plane, concentric bright and dark rings appear in the image. The location of the rings can be related to cell topography, and the ring contrast can be correlated to the stiffness and density of the cell. In addition, the character of the images of single cells varies dramatically when the substratum upon which they are grown is changed to a different material. By careful selection of the substratum, the information content of the acoustic images can be increased. Our analysis of acoustic images of actively motile cells indicates that leading lamella are less dense or stiff than the quiescent trailing processes of the cells. Images PMID:6940179

Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

This paper is a review of our results of the application of atomic force microscopy (AFM) to the three-dimensional observation of living cells. First, we showed AFM images of living cultured cells in fluid. Contact mode AFM of living cells provided precise information on the shape of cellular processes (such as spike-like processes or lamellipodia) at the cellular margin. The contour of cytoskeletal elements just beneath the cell membrane was also clearly observable on the upper surface of the cells. Secondly, we showed the data on the discrepancy between the AFM images of living cells and fixed cells. These findings were useful for evaluating AFM images of living cells. Finally, we described the time-lapse AFM of living cells. A fluid chamber system enabled us to obtain AFM images of living cells for over 1 h at time intervals of 2-4 min. A series of these AFM images were useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movies produced by sequential AFM images also gave a realistic view of the cellular dynamics.

Confocal microscopy is an imaging paradigm that allows optical sectioning of almost any material with increased axial and lateral spatial resolution and better image contrast. We have applied this technology to the study of the living eye of cats, albino rabbits, and humans. The technique allows in vivo, noninvasive, real time images of the eye at magnifications (630x) which allow resolution of anatomical detail at the cellular level. In this paper we report details of our current instrument techniques and some of our results. The past development, present state-of-the-art, and projected future advances and applications of this novel microscopy are discussed. Preliminary observations are reported for all layers of the cornea, the limbus, and wound-healing responses in single animals. PMID:2407380

Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:9251784

Summary Intravital microscopy (IVM) is a powerful tool that enables imaging various biological processes in live animals. Here, we describe a series of procedures designed to image subcellular structures, such as endsosomes and secretory vesicles in the salivary glands (SGs) of live rats. To this aim, we used fluorescently labeled molecules and/or fluorescently-tagged proteins that were transiently transfected in the live animal. PMID:23027003

Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. PMID:23042524

Real-time, compact x-ray microscopy has the potential to benefit many scientific fields, including microbiology, pharmacology, organic chemistry, and physics. Single frame x-ray micro-radiography, produced by a compact, solid-state laser plasma source, allows scientists to use x-ray emission for elemental analysis, and to observe biological specimens in their natural state. In this study, x-ray images of mouse kidney tissue, live bacteria, Pseudomonas aeruginosa and Burkholderia cepacia, and the bacteria's interaction with the antibiotic gentamicin, are examined using x-ray microscopy. For the purposes of comparing between confocal microscopy and x-ray microscopy, we introduced to our work the technique of gold labeling. Indirect immunofluorescence staining and immuno-gold labeling were applied on human lymphocytes and human tumor cells. Differential interference contrast microscopy (DIC) showed the lymphocyte body and nucleus, as did x-ray microscopy. However, the high resolution of x-ray microscopy allows us to differentiate between the gold particles bound to the antibodies and the free gold. A compact, tabletop Nd: glass laser is used in this study to produce x-rays from an Yttrium target. An atomic force microscope is used to scan the x-ray images from the developed photo-resist. The use of compact, tabletop laser plasma sources, in conjunction with x-ray microscopy, is a new technique that has great potential as a flexible, user-friendly scientific research tool.

The development of intravital microscopy has provided unprecedented capacity to study the tumor microenvironment in live mice. The dynamic behavior of cancer, stromal, vascular, and immune cells can be monitored in real time, in situ, in both primary tumors and metastatic lesions, allowing treatment responses to be observed at single cell resolution and therapies tracked in vivo. These features provide a unique opportunity to elucidate the cellular mechanisms underlying the biology and treatment of cancer. We describe here a method for imaging the microenvironment of subcutaneous tumors grown in mice using intravital microscopy. PMID:27581025

Scanned probe microscopy is a general term encompassing the science of imaging based on piezoelectric driven probes for measuring local changes in nanoscale properties of materials and devices. Techniques like scanning tunneling microscopy, atomic force microscopy, and scanning potentiometry are becoming common tools in the production and development labs in the semiconductor industry. The author presents several examples of applications specific to the development of high temperature superconducting thin films and thin-film devices.

For more than a decade MIT's George R. Harrison Spectroscopy Laboratory has been developing quantitative phase microscopy (QPM) for biological study. Measurements of a point field were made in the mid 90s, then extended to the full 2D field, and recently, to 3D by using tomography. In the first part of this thesis improvements in the techniques of Fourier Phase Microscopy (FPM) and Hilbert Phase Microscopy (HPM) and their applications to characterize cells and tissues are reported. Tomographic phase microscopy (TPM) provides quantitative information and highly detailed structural information about a live cell, but in its current form it can only examine one cell at a time. Many biological applications including statistical analysis of a large collection of cells such as flow cytometry need a tomography technique that can measure many cells at a time. For the second part of this thesis we have developed a new tomography technique that can measure many cells continuously. In this study we demonstrate the new technique by translating a live cell across a focused beam. This beam is composed of many angular plane waves, and by applying a so-called synthetic aperture algorithm we retrieve individual wave components of the focused beam. We demonstrate for the first time that we can retrieve the field of the focused beam and synthesize any arbitrary angular plane wave. We then construct a 3D map of the variations of the refractive index in a live cell from a series of these synthesized angular plane waves. This new technique is the first step needed to analyze cells flowing through a beam to provide a high-throughput 3D refractive index tomograms that can be used as a new kind of statistical optical assay of living cells.

Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

We have used the inherent surface sensitivity of second harmonic generation to develop an instrument for nonlinear optical microscopy of surfaces and interfaces. We have demonstrated the use of several nonlinear optical responses for imaging thin films. The second harmonic response of a thin film of C{sub 60} has been used to image patterned films. Two photon absorption light induced fluorescence has been used to image patterned thin films of Rhodamine 6G. Applications of nonlinear optical microscopy include the imaging of charge injection and photoinduced charge transfer between layers in semiconductor heterojunction devices as well as across membranes in biological systems.

Here we report on the use of atomic force microscopy (AFM) to study surface oscillations coming from internal live processes of insects. With a specially designed AFM stage to keep an insect motion partially restricted, the AFM can record internal oscillations on different parts of the insect. We demonstrate the method for a fly, mosquito, and lady beetle. We show that AFM can provide information about the spectral behavior that has not been studied so far, 10-600 Hz range, detecting amplitudes down to subnanometer level.

A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a "3-D cluster" as well as subcellular organelles like the nucleoli and local internal refractive index changes. PMID:24723114

Quantitative analysis of biological image data generally involves the detection of many pixel spots. In live mitochondria video image, for which fluorescent microscopy is often used, the signal-to-noise ratio (SNR) can be extremely low, making the detection and tracking of mitochondria particle difficult. It is especially not easy to get the movement curve when the movement of the mitochondria involves its self-move and the motion caused by the neuron. An tracking algorithm for live mitochondria is proposed in this paper. First the whole image sequence is frame-to-frame registered, in which the edge corners are chosen to be the feature points. Then the mitochondria particles are tracked by frame-to-frame displacement vector. The algorithm proposed has been applied to the dynamic image sequence including neuron and mitochondria, saving time without manually picking up the feature points. It provides an new method and reference for medical image processing and biotechnological research. PMID:22616189

The characterization of the surface of selected space-exposed polymer films by Scanning Tunneling Microscopy (STM) is reported. Principles of STM, an emerging new technique for materials analysis, are reviewed. The analysis of several films which received up to 5.8 years of low Earth orbital (LEO) exposure onboard the NASA Long Duration Exposure Facility (LDEF) is discussed. Specimens included FEP Teflon thermal blanket material, Kapton film, and several experimental polymer films. Ultraviolet and atomic oxygen-induced crazing and erosion are described. The intent of this paper is to demonstrate how STM is enhancing the understanding of LEO space environmental effects on polymer films.

We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities. PMID:27523472

Recently, the existence of multiple micro-domains of extracellular potential around individual cells have been revealed by voltage reporter dye using fluorescence microscopy. One hypothesis is that these long lasting potential patterns play a vital role in regulating important cell activities such as embryonic patterning, regenerative repair and reduction of cancerous disorganization. We used multifunctional Scanning Ion Conductance Microscopy (SICM) to study these extracellular potential patterns of single cell with higher spatial resolution. To validate this novel technique, we compared the extracellular potential distribution on the fixed HeLa cell surface and Polydimethylsiloxane (PDMS) surface and found significant difference. We then measured the extracellular potential distributions of living melanocytes and melanoma cells and found both the mean magnitude and spatial variation of extracellular potential of the melanoma cells are bigger than those of melanocytes. As compared to the voltage reporter dye based fluorescence microscope method, SICM can achieve quantitative potential measurements of non-labeled living cell membranes with higher spatial resolution.

Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell's elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM. PMID:26011471

Atomic-force microscopy is used to study the supramolecular structure of submicron films of electroactive thermally stable polymer (polydiphenylenephthalide (PDP)). It has been demonstrated that PDP films produced using centrifuging are solid homogeneous films with thicknesses down to several nanometers, which correspond to two or three monomolecular layers. The film volume is structurized at thicknesses greater than 100 nm. The study of the rheological properties of solutions used for film production yields a crossover point that separates the domains of strongly diluted and semidiluted solutions. A transition from the globular structure to the associate structure is observed in films that are produced using solutions with a boundary concentration. A model of the formation of polymer film that involves the presence of associates in the original solution is discussed.

Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

We present the observation of living specimens with subwavelength resolution by using the nonoptically probing near-field microscopy we have developed recently. In the near-field microscope, the optical field distributions near the specimens are recorded as the surface topography of a photosensitive film, and the topographical distributions are readout with an atomic-force microscopy. Since the near-field microscope does not require the scanning of a probe tip for illumination or detection or scattering of light, it is possible to observe moving biological specimens and fast phenomena. We demonstrate the observation of a moving paramecium and euglena gracilis with subwavelength resolution. The observation of the nucleus inside a euglena cell was also demonstrated.

We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives. PMID:25265458

Bacterial film (biofilm) microbes exist within spatial (nutrient, electron-acceptor, pH, etc.) gradients of their own making. Correspondingly, biofilm bacteria are physiologically and functionally distinct from free-floating bacteria and from their own species at differing biofilm depths. This article describes our efforts to develop noninvasive nuclear magnetic resonance (NMR) technologies for biofilm-metabolism studies. This involves integrating NMR with controlled-cultivation methods to interrogate microbial physiology live and under known growth conditions. NMR is uniquely capable of providing depth-resolved metabolic and transport information in a non-invasive, non-sample-consuming fashion, providing information required for experimental reactive transport studies. We have studied mono-species biofilms relevant to environment remediation and human health. We describe these technologies, discuss their advantages and limitations, and give examples of their application.

Pulmonary surfactant facilitates breathing by forming a surface tension reducing film at the air-liquid interface of the alveoli. The objective was to characterize the structure of surfactant films using endogenous rat surfactant. Solid-support surfactant films, at different surface pressures, were obtained using a Langmuir balance and were analyzed using atomic force microscopy. The results showed a lipid film structure with three distinct phases: liquid expanded, liquid ordered and liquid condensed. The area covered by the liquid condensed domains increased as surface pressure increased. The presence of liquid ordered phase within these structures correlated with the cholesterol content. At a surface pressure of 50 mN/m, stacks of bilayers appeared. Several structural details of these films differ from previous observations made with goat and exogenous surfactants. Overall, the data indicate that surfactant films demonstrate phase separation at low surface pressures and multilayer formation at higher pressure, features likely important for normal surfactant function. PMID:21704443

The microstructure of 0022-3727/31/14/005/img11Cu0022-3727/31/14/005/img12 bilayer film grown on 0022-3727/31/14/005/img13 substrate was studied by high-resolution transmission electron microscopy (HREM). The results showed that the 0022-3727/31/14/005/img14 film is epitaxially grown on the 0022-3727/31/14/005/img13 substrate with c axis orientation. Planar defects, grain boundaries, moiré patterns, a axis oriented 0022-3727/31/14/005/img14 and impurity particulates are found in the 0022-3727/31/14/005/img14 film. The 0022-3727/31/14/005/img18 film was grown on the 0022-3727/31/14/005/img14 film with a columnar structure. However, some region of the 0022-3727/31/14/005/img18 film is single crystalline, but with strain bands. The development of strain bands in the 0022-3727/31/14/005/img18 film could be a result of lattice mismatch between 0022-3727/31/14/005/img14 and 0022-3727/31/14/005/img18 films and the surface roughness of the 0022-3727/31/14/005/img14 film. In consequence, the dielectric properties of the strained STO film are greatly decreased compared to the bulk single crystalline STO.

In this report, we report on progress achieved from 12/1/92 to 10/1/93 under the grant entitled 'Scanning Tunneling Microscopy Studies of Diamond Films and Optoelectronic Materials'. We have set-up a chemical vapor deposition (CVD) diamond film growth system and a Raman spectroscopy system to study the nucleation and growth of diamond films with atomic resolution using scanning tunneling microscopy (STM). A unique feature of the diamond film growth system is that diamond films can be transferred directly to the ultrahigh vacuum (UHV) chamber of a scanning tunneling microscope without contaminating the films by exposure to air. The University of North Texas (UNT) provided $20,000 this year as matching funds for the NASA grant to purchase the diamond growth system. In addition, UNT provided a Coherent Innova 90S Argon ion laser, a Spex 1404 double spectrometer, and a Newport optical table costing $90,000 to set-up the Raman spectroscopy system. The CVD diamond growth system and Raman spectroscopy system will be used to grow and characterize diamond films with atomic resolution using STM as described in our proposal. One full-time graduate student and one full-time undergraduate student are supported under this grant. In addition, several graduate and undergraduate students were supported during the summer to assist in setting-up the diamond growth and Raman spectroscopy systems. We have obtained research results concerning STM of the structural and electronic properties of CVD grown diamond films, and STM and scanning tunneling spectroscopy of carbon nanotubes. In collaboration with the transmission electron microscopy (TEM) group at UNT, we have also obtained results concerning the optoelectronic material siloxene. These results were published in refereed scientific journals, submitted for publication, and presented as invited and contributed talks at scientific conferences.

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

The structure of Fe1 + δTe1 - x Se x films ( x = 0; 0.05) grown on single-crystal MgO and LaAlO3 substrates has been investigated by transmission and scanning transmission electron microscopy. The study of Fe1.11Te/MgO structures has revealed two crystallographic orientation relationships between the film and substrate. It is shown that the lattice mismatch between the film and substrate is compensated for by the formation of misfit dislocations. The Burgers vector projection is determined. The stresses in the film can partially be compensated for due to the formation of an intermediate disordered layer. It is shown that a FeTe0.5Se0.5 film grown on a LaAlO3 substrate is single-crystal and that the FeTe0.5Se0.5/LaAlO3 interface in a selected region is coherent. The orientation relationships between the film and substrate are also determined for this case.

The structure of Fe{sub 1+δ}Te{sub 1−x}Se{sub x} films (x = 0; 0.05) grown on single-crystal MgO and LaAlO{sub 3} substrates has been investigated by transmission and scanning transmission electron microscopy. The study of Fe{sub 1.11}Te/MgO structures has revealed two crystallographic orientation relationships between the film and substrate. It is shown that the lattice mismatch between the film and substrate is compensated for by the formation of misfit dislocations. The Burgers vector projection is determined. The stresses in the film can partially be compensated for due to the formation of an intermediate disordered layer. It is shown that a FeTe{sub 0.5}Se{sub 0.5} film grown on a LaAlO{sub 3} substrate is single-crystal and that the FeTe{sub 0.5}Se{sub 0.5}/LaAlO{sub 3} interface in a selected region is coherent. The orientation relationships between the film and substrate are also determined for this case.

Nuclear magnetic resonance (NMR) is a fundamental research tool that is widely used in many fields. Despite its powerful applications, unfortunately the low sensitivity of conventional NMR makes it difficult to study thin film or nano-sized samples. In this work, we report the first NMR spectrum obtained from general thin films by using magnetic resonance force microscopy (MRFM). To minimize the amount of imaging information inevitably mixed into the signal when a gradient field is used, we adopted a large magnet with a flat end with a diameter of 336 μm that generates a homogeneous field on the sample plane and a field gradient in a direction perpendicular to the plane. Cyclic adiabatic inversion was used in conjunction with periodic phase inversion of the frequency shift to maximize the SNR. In this way, we obtained the 19F NMR spectrum for a 34 nm-thick CaF2 thin film. PMID:24217000

We introduce an alternative type of probe for scanning tunneling microscopy (STM). Instead of using a needle-like tip made from a piece of metallic wire, a sharp-edged cleaved insulating substrate, which is initially covered by a thin conductive film, is used. The sharp tip is formed at the intersection of the two cleaved sides. Using this approach a variety of materials for STM probes can be used, and functionalization of STM probes is possible. The working principle of different probes made of metallic (Pt, Co, and CoB), indium-tin oxide, as well as Cu/Pt and Co/Pt multilayer films are demonstrated by STM imaging of clean Cu(001) and Cu(111) surfaces as well as the epitaxial Co clusters on Cu(111). PMID:26636763

High Resolution Transmission Electron Microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by non-specialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods was steadily improved reaching now into the sub Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this paper, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this paper is the proposal of a reproducible method for film thickness determination.

Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. Images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter-based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes. PMID:24077519

Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.

Here we describe methods that are useful for exploring the formation and functions of primary cilia in living cells. First we describe multiple protocols for visualizing solitary cilia that extend away from the cell body. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The second protocol describes how to quantify movement of fluorescent molecules within the cilium. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission. PMID:23208547

Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. PMID:26133185

Silicon carbide (SiC) holds great potential as an electronic material because of its wide band gap energy, high breakdown electric field, thermal stability, and resistance to radiation damage. Possible aerospace applications of high-temperature, high-power, or high-radiation SiC electronic devices include sensors, control electronics, and power electronics that can operate at temperatures up to 600 C and beyond. Commercially available SiC devices now include blue light-emitting diodes (LED's) and high-voltage diodes for operation up to 350 C, with other devices under development. At present, morphological defects in epitaxially grown SiC films limit their use in device applications. Research geared toward reducing the number of structural inhomogeneities can benefit from an understanding of the type and nature of problems that cause defects. The Atomic Force Microscope (AFM) has proven to be a useful tool in characterizing defects present on the surface of SiC epitaxial films. The in-house High-Temperature Integrated Electronics and Sensors (HTIES) Program at the NASA Lewis Research Center not only extended the dopant concentration range achievable in epitaxial SiC films, but it reduced the concentration of some types of defects. Advanced structural characterization using the AFM was warranted to identify the type and structure of the remaining film defects and morphological inhomogeneities. The AFM can give quantitative information on surface topography down to molecular scales. Acquired, in part, in support of the Advanced High Temperature Engine Materials Technology Program (HITEMP), the AFM had been used previously to detect partial fiber debonding in composite material cross sections. Atomic force microscopy examination of epitaxial SiC film surfaces revealed molecular-scale details of some unwanted surface features. Growth pits propagating from defects in the substrate, and hillocks due, presumably, to existing screw dislocations in the substrates, were

The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

Gold nanoparticle films are of interest in several branches of science and technology, and accurate sample characterization is needed but technically demanding. We prepared such films by DC magnetron sputtering and recorded their mass thickness by Rutherford backscattering spectroscopy. The geometric thickness d{sub g}—from the substrate to the tops of the nanoparticles—was obtained by scanning electron microscopy (SEM) combined with image analysis as well as by atomic force microscopy (AFM). The various techniques yielded an internally consistent characterization of the films. In particular, very similar results for d{sub g} were obtained by SEM with image analysis and by AFM.

We present a summary of the research, citations of publications resulting from the research and abstracts of such publications. We have made no inventions in the performance of the work in this project. The main goals of the project were to set up a Chemical Vapor Deposition (CVD) diamond growth system attached to an UltraHigh Vacuum (UHV) atomic resolution Scanning Tunneling Microscopy (STM) system and carry out experiments aimed at studying the properties and growth of diamond films using atomic resolution UHV STM. We successfully achieved these goals. We observed, for the first time, the atomic structure of the surface of CVD grown epitaxial diamond (100) films using UHV STM. We studied the effects of atomic hydrogen on the CVD diamond growth process. We studied the electronic properties of the diamond (100) (2x1) surface, and the effect of alkali metal adsorbates such as Cs on the work function of this surface using UHV STM spectroscopy techniques. We also studied, using STM, new electronic materials such as carbon nanotubes and gold nanostructures. This work resulted in four publications in refereed scientific journals and five publications in refereed conference proceedings.

Summary The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts, and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. PMID:22396229

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices. PMID:22098754

Fluorescence microscopy (FM) is a powerful tool for observing specific molecular components in living cells, but its spatial resolution is relatively low. In contrast, electron microscopy (EM) provides high-resolution information about cellular structures, but it cannot provide temporal information in living cells. To achieve molecular selectivity in imaging at high resolution, a method combining EM imaging with live-cell fluorescence imaging, known as live correlative light-EM (CLEM), has been developed. In this method, living cells are first observed by FM, fixed in situ during the live observation and then subjected to EM observation. Various fluorescence techniques and tools can be applied for FM, resulting in the generation of various modified methods that are useful for understanding cellular structure in high resolution. Here, we review the methods of CLEM and live-cell imaging associated with CLEM (live CLEM). Such methods can greatly advance the understanding of the function of cellular structures on a molecular level, and thus are useful for medical fields as well as for basic biology. PMID:27385786

Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking. PMID:27145353

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". As a unique tool for biological discovery, this platform has been applied to

Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our livemicroscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity. PMID:22028887

The structural and functional heterogeneity of the kidney ensures a diversity of response in health and disease. Multiphoton microscopy has improved our understanding of kidney physiology and pathophysiology by enabling the visualization of the living kidney in comparison with the static view of previous technologies. The use of multiphoton microscopy with rodent models in conjunction with endogenous fluorescence and exogenous infused dyes permits the measurement of renal processes, such as glomerular permeability, juxtaglomerular apparatus function, tubulointerstitial function, tubulovascular interactions, vascular flow rate, and the intrarenal renin-angiotensin-aldosterone system. Subcellular processes, including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes apoptosis and necrosis, can also be measured by multiphoton microscopy. This has allowed valuable insight into the pathophysiology of diabetic nephropathy, renal ischemia-reperfusion injury, hypertensive nephropathy, as well as inflammatory responses of the kidney. The current review presents an overview of multiphoton microscopy with a focus on techniques for imaging the kidney and gives examples of instances where multiphoton microscopy has been utilized to study renal pathophysiology in the living kidney. With continued advancements in the field of biological optics and increased adoption in experimental nephrology, multiphoton microscopy will undoubtedly continue to create new paradigms in kidney disease. PMID:24525825

Within the lateral organisation of plasma membranes of polarized cell types there exist heterogenous microdomains of distinct lipid composition, the small size of which (10–200 nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes. PMID:21126508

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations. PMID:27259929

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

The photothermal detection technique is an innovative and non-contact method to investigate the properties of films on workpieces. This paper describes a novel experimental set-up for thickness microscopy based on photothermal radiometry. The correlation between the thermal wave signal and the film thickness is deduced and evaluated to determine the film thickness with a lateral resolution of less than 1mm. Results indicate that the thickness microscopy is a useful method to characterize thin films and has the potential to be applied in-process. PMID:19046925

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes. PMID:27515076

Localized surface plasmon enhanced microscopy based on nanoislands of random spatial distribution was demonstrated for imaging live cells and molecular interactions. Nanoislands were produced without lithography by high temperature annealing under various processing conditions. The localization of near-field distribution that is associated with localized surface plasmon on metallic random nanoislands was analyzed theoretically and experimentally in comparison with periodic nanostructures. For experimental validation in live cell imaging, mouse macrophage-like cell line stained with Alexa Fluor 488 was prepared on nanoislands. The results suggest the possibility of attaining the imaging resolution on the order of 80 nm.

The authors classify good, leaky, and broken field effect transistors (FET's) in a live 90nm flip-chip microprocessor using functional infrared emission spectral microscopy. The FET's are in the active layer that is sandwiched between a thick heat-absorbing silicon material and a highly reflecting grid of metal interconnects. Together they are optically imaged only as a single bright blob. They classify FET's individually from their distinct electroluminescence spectra that are recovered efficiently by spectral decomposition of the detected composite spectrum. Leaky FET's have no apparent structural damage and are detectable only in live microprocessors.

Living cells are active mechanical structures which evolve within and in response to their local microenvironments. Various cell types possess different mechanical properties and respond uniquely to growth, environmental changes, and the application of chemical stimuli. Here we present a powerful approach which combines high resolution Atomic Force Microscopy with Fluorescence Microscopy to systematically obtain real-time micrometer and sub-micrometer resolution elasticity maps for live neuronal cells cultured on glass substrates. Through this approach we measure the topography, the elastic properties, and the dynamics of neuronal cells, and identify changes in cytoskeletal components during axonal growth, chemical modification, and changes in ambient temperature. We will also show high resolution elasticity measurements of the cell body and of axons/dendrites during growth, as well as identification of cytoskeletal components during cell growth and environmental changes.

Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

We present a microfluidic system for time-lapsed, live cell microscopy with the ability to control solution exchange via a dynamic flow controller. The application specific microfluidic plates are designed to maintain adherent and non-adherent cell types for multiple days with continuous medium perfusion. Upstream channels with flow controlled via custom software allow the delivery of unique exposure profiles to the cultured cells, such as square waves, step functions, ramps, etc. PMID:19209350

Live cell microscopy using fluorescent proteins and small fluorescent probes is a well-established and essential tool for cell biology; however, there is a considerable need for noninvasive techniques able to study tissue and cell dynamics without the need to introduce chemical or genetically encoded probes. Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging tool for cell biologists to examine live cell dynamics with chemical specificity in a label-free, noninvasive way. CARS is a multiphoton process offering intrinsic three-dimensional submicron resolution, where the image contrast is obtained from light inelastically scattered by the vibrations of endogenous chemical bonds. CARS is particularly well suited to study lipid biology, since the CARS signal of localized lipids (exhibiting a large amount of identical bonds in the focal volume) is very strong. Conversely, photostable, lipid-specific markers for fluorescence microscopy are difficult to produce and the process of labeling often affects lipid localization and function, making imaging lipids in live cells challenging, and accurate quantification often impossible. Here, we describe in detail the principles behind our experimental setup for performing CARS microscopy of lipid droplets on live cells. Since typical vibrational resonances in liquid have coherence times in the picosecond range, CARS is preferably implemented with picosecond lasers which are however expensive and less efficient than femtosecond lasers, which could also be used for other multiphoton techniques such as two-photon fluorescence. In our setup, we show that femtosecond lasers can be spectrally focused in a simple, alignment insensitive, and cost-effective way to achieve a vibrational excitation similar to picosecond lasers. This opens the way to integrate CARS and two-photon fluorescence in a single multimodal instrument for its widespread application. We also describe our dual frequency CARS system which eliminates

A study has been made of the passive film remaining over pits on stainless steel using a high resolution transmission electron microscope. Type 305 stainless steel was passivated in a borate buffer solution and pitted in ferric chloride. Passive films formed at 0.2 V relative to a saturated calomel electrode were found to be amorphous. Films formed at higher potentials showed only broad diffraction rings. The passive film was found to cover a remnant lacy structure formed over pits passivated at 0.8 V. The metallic strands of the lace were roughly hemitubular in shape with the curved surface facing the center of the pit.

The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open-access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze polarized fluorescence. PMID:26061244

The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open-access software (www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze polarized fluorescence. PMID:26061244

Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization. PMID:23895420

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells. PMID:23762252

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells. PMID:23762252

The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

Ultrahigh-speed microscopy of living specimens requires ultrabright illumination. Moreover, the duration of illumination should be sufficiently long, on the order of at least several tens of milliseconds, in order to investigate the dynamic state of living specimens. However, specimens are exposed to a high risk of damage by the intense illumination. The brightness and pulse duration of illumination have to be continuously controlled for use in the ultrahigh-speed microscopy of living specimens. Commercial or laboratory-made illumination systems do not satisfy the abovementioned requirements. In this paper, the development of a bright and long-pulse illumination system for ultrahigh-speed microscopy of living specimens is presented. A xenon flashlamp with an arc length of 1.5 mm has been used as the light source. The electrical power supply consists of a voltage-regulated circuit, a capacitor bank, and a control circuit including an insulated-gate bipolar transistor as a gating device, which provides a large rectangular current pulse with the duration in the range to the order of several tens of milliseconds. The brightness, pulse duration, and repetition rate can be easily and continuously controlled. The illumination developed in the present study is installed in an inverted fluorescence microscope equipped with a high-speed camera in order to evaluate the performance as an illumination source. A fluorescent image of the living spermatozoa of a mouse obtained at a frame rate of 8 kHz shows good contrast. Such an image cannot be obtained using a commercial illumination system.

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criteria. Indeed, first we will focus on those studies in which organs where imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures. PMID:20372919

The research work in this letter is on the microtribological properties of poly(ether ketone ketone) (PEKK) and sulfonated PEKK (S-PEKK) thin films. Polystyrene (PS) was used as a reference for the investigation. Atomic-force-microscopy (AFM) techniques were used for observing the topography and friction properties of the macromolecular thin films at the nanometer scale. The polymeric thin films were fabricated by spin coating at a speed of 4000 rotations per minute (rpm).

Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view. PMID:27562163

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm-2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm-2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. PMID:26481189

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. PMID:26481189

Wide-field fluorescence microscopy was used to monitor the co-localization of the homeodomain (HD) transcription factor Pit-1 and the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPa), each labeled with fluorescent proteins (FP) in the living cell nucleus. Fluorescence resonance energy transfer (FRET) microscopy was used to resolve the angstrom-scale spatial relationships of these expressed proteins, and the effect of a Pit-1 point mutation on the interaction with C/EBPa was characterized. Two-photon excitation fluorescence lifetime imaging microscopy (2p-FLIM) was then used as an independent method to detect these protein interactions. The excited-state lifetime for the cyan FP (CFP) labeling C/EBPa was determined, and the measurements were repeated in cells co-expressing yellow FP (YFP) labeled-proteins. The CFP lifetime was decreased in the presence of the YFP acceptor, which is consistent with donor quenching by FRET. This was verified by acceptor photobleaching, which caused a shift in the donor lifetime to that similar to the donor alone. However, a significant limitation of this technique was demonstrated by the observation that high-energy 2p-excitation resulted in CFP photobleaching and a parallel decrease in its excited-state lifetime. The key question is whether the sensitivity of this imaging approach will be sufficient to acquire significant data from living cells expressing physiological levels of the labeled proteins.

Cell morphology is the research foundation in many applications related to the estimation of cell status, drug response, and toxicity screening. In biomedical field, the quantitative phase detection is an inevitable trend for living cells. In this paper, the morphological change of HeLa cells treated with methanol of different concentrations is detected using digital holographic microscopy. The compact image-plane digital holographic system is designed based on fiber elements. The quantitative phase image of living cells is obtained in combination with numerical analysis. The statistical analysis shows that the area and average optical thickness of HeLa cells treated with 12.5% or 25% methanol reduce significantly, which indicates that the methanol with lower concentration could cause cellular shrinkage. The area of HeLa cells treated with 50% methanol is similar to that of normal cells (P > 0.05), which reveals the fixative effect of methanol with higher concentration. The maximum optical thickness of the cells treated with 12.5%, 25%, and 50% methanol is greater than that of untreated cells, which implies the pyknosis of HeLa cells under the effect of methanol. All of the results demonstrate that digital holographic microscopy has supplied a noninvasive imaging alternative to measure the morphological change of label-free living cells. PMID:23424605

A new method to visualize the large-scale crystal grain morphology of organic polycrystalline films is proposed. First, optical anisotropic transmittance images of polycrystalline zinc phthalocyanine (ZnPc) films vacuum deposited by weak epitaxial growth (WEG) method were acquired with polarized optical microscopy (POM). Then morphology properties including crystal grain size, distribution, relative orientation, and crystallinity were derived from these images by fitting with a transition dipole model. At last, atomic force microscopy (AFM) imaging was carried out to confirm the fitting and serve as absolute references. This method can be readily generalized to other organic polycrystalline films, thus providing an efficient way to access the large-scale morphologic properties of organic polycrystalline films, which may prove to be useful in industry as a film quality monitoring method. Project supported by the National Natural Science Foundation of China (Grant No. 20933010) and the National Basic Research Program of China (Grant No. 2013CB834800).

Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues.

Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer. PMID:12756303

Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues. PMID:24336094

We propose using the method of holographic microscopy to detect fine morphologic changes in living cells. An "LSM 510" laser confocal scanning microscope is modified to allow recording digital microholograms which can be used to reconstruct the amplitude and phase of the radiation transmitting through the sample. Measuring the phase increment of the object beam in cells and the intercellular space yields information on the optical length of the ray path in the cells (spatial dimensions and the refractive index), which in turn contains information on changes in the morphology and intracellular contents. Calcium activity is studied by means of fluorescent microscopy which makes it possible to detect minor variations in the intracellular concentration of calcium ions. By studying the dynamics of calcium oscillations and variations in the optical thickness, conclusions are made about the interrelation of functional and morphological variations, and comparative analysis of these variations is performed.

Analysis of cellular pathways requires concentration measurements of dynamically interacting molecules within the three-dimensional (3D) space of single living cells. Förster resonance energy transfer (FRET) microscopy from widefield, from confocal, and potentially from superresolution microscopes can access this information; however, these measurements are distorted by the inherent 3D blurring of optical imaging, spectral overlap of fluorophores, and detection noise. We propose a mathematical model of these processes and demonstrate, through simulation, how these distortions limit the dynamic range and sensitivity of conventional FRET microscopy. Using this model, we devise and validate a new approach (called 3D-FRET stoichiometry reconstruction, 3DFSR) for reconstructing 3D distributions of bound and free fluorescent molecules. Previous attempts to reconstruct 3D-FRET data relied on sequential spectral unmixing and deconvolution, a process that corrupts the detection statistics. We demonstrate that 3DFSR is superior to these approaches since it simultaneously models spectral mixing, optical blurring, and detection noise. To achieve the full potential of this technique, we developed an instrument capable of acquiring 3D-FRET data rapidly and sensitively from single living cells. Compared with conventional FRET microscopy, our 3D-FRET reconstruction technique and new instrumentation provides orders of magnitude gains in both sensitivity and accuracy wherein sustained high-resolution four-dimensional (x,y,z,t) imaging of molecular interactions inside living cells was achieved. These results verify previous observations that Cdc42 signaling is localized to the advancing margins of forming phagosomes in macrophages. PMID:18339754

Abstract A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm. PMID:26844929

Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

Atomic force microscopy (AFM) is a powerful and versatile tool for probing the mechanical properties of biological samples. This chapter describes the procedures for using AFM indentation to measure the elastic moduli of living cells. We include step-by-step instructions for cantilever calibration and data acquisition using a combined AFM/optical microscope system, as well as a detailed protocol for data analysis. Our protocol is written specifically for the BioScope™ Catalyst™ AFM system (Bruker AXS Inc.); however, most of the general concepts can be readily translated to other commercial systems. PMID:23027009

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm. PMID:26844929

Semitransparent porous silicon substrates have been developed for pairing nanostructure-initiator mass spectrometry (NIMS) imaging with traditional optical-based microscopy techniques. Substrates were optimized to generate the largest NIMS signal while maintaining sufficient transparency to allow visible light to pass through for optical microscopy. Using these substrates, both phase-contrast and NIMS images of phospholipids from a scratch-wounded cell monolayer were obtained. NIMS images were generated using a spatial resolution of 14 μm. Coupled with further improvements in spatial resolution, this approach may allow for the localization of intact biological molecules within cells without the need for labeling. PMID:23146026

Scanning probe microscopy (SPM) equipped in high temperature nanoindentation instrument is adopted to in situ characterize the oxide film growth on Ni-base single crystal at nanoscale. SPM images reveal a transition of oxide film configuration that the originally flat surface roughens during oxidation. Based on the stress-diffusion coupling effect during oxidation, the stress evolution in the oxide film and the evolution of surface configuration are analyzed. A new method to infer the critical stress in the oxide film at the transition point is proposed by measuring the undulated surface wavelength based on the surface morphology obtained by SPM.

Quantitative phase microscopy is applied to image temporal changes in the refractive index (RI) distributions of solutions created by microbicidal films undergoing hydration. We present a novel method of using an engineered polydimethylsiloxane structure as a static phase reference to facilitate calibration of the absolute RI across the entire field. We present a study of dynamic structural changes in microbicidal films during hydration and subsequent dissolution. With assumptions about the smoothness of the phase changes induced by these films, we calculate absolute changes in the percentage of film in regions across the field of view.

ZnTe thin films obtained by the electron beam evaporation technique were subjected to thermal annealing at 500 Degree-Sign C for 2 hours. The as deposited films were amorphous but transformed to the crystalline state under influence of the thermal treatment. There is increase in optical absorption due to the heat treatment caused by increase in free carrier concentration. Conductive atomic force microscopy shows the presence of electronic inhomogeneities in the films. This is attributed to local compositional variations in the films. I-V analysis in these systems indicates formation of Schottky junction at the metal semiconductor (M-S) interface.

An outdoor soil burial test was carried out to evaluate the degradation of commercially available LDPE carrier bags in natural soil for up to 2 years. Biodegradability of low density polyethylene films in soil was monitored using both optical and scanning electron microscopy (SEM). After 7-9 months of soil exposure, microbial colonization was evident on the film surface. Exposed LDPE samples exhibit progressive changes towards degradation after 17-22 months. SEM images reveal signs of degradation such as exfoliation and formation of cracks on film leading to disintegration. The possible degradation mode and consequences on the use and disposal of LDPE films is discussed. PMID:20207547

Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET ). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ~2 d. PMID:21886099

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse-chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial-temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse-chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

Stiffness of live cardiomyocytes isolated from control and diabetic mice was measured using the atomic force microscopy nanoindentation method. Type 1 diabetes was induced in mice by streptozotocin administration. Histological images of myocardium from mice that were diabetic for 3 mo showed disorderly lineup of myocardial cells, irregularly sized cell nuclei, and fragmented and disordered myocardial fibers with interstitial collagen accumulation. Phalloidin-stained cardiomyocytes isolated from diabetic mice showed altered (i.e., more irregular and diffuse) actin filament organization compared with cardiomyocytes from control mice. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) pump expression was reduced in homogenates obtained from the left ventricle of diabetic animals compared with age-matched controls. The apparent elastic modulus (AEM) for live control or diabetic isolated cardiomyocytes was measured using the atomic force microscopy nanoindentation method in Tyrode buffer solution containing 1.8 mM Ca(2+) and 5.4 mM KCl (physiological condition), 100 nM Ca(2+) and 5.4 mM KCl (low extracellular Ca(2+) condition), or 1.8 mM Ca(2+) and 140 mM KCl (contraction condition). In the physiological condition, the mean AEM was 112% higher for live diabetic than control isolated cardiomyocytes (91 ± 14 vs. 43 ± 7 kPa). The AEM was also significantly higher in diabetic than control cardiomyocytes in the low extracellular Ca(2+) and contraction conditions. These findings suggest that the material properties of live cardiomyocytes were affected by diabetes, resulting in stiffer cells, which very likely contribute to high diastolic LV stiffness, which has been observed in vivo in some diabetes mellitus patients. PMID:25163520

Atomic force microscopy (AFM) allows the acquisition of high-resolution images and the measurement of mechanical properties of living cells under physiological conditions. AFM cantilevers with blunted pyramidal tips are commonly used to obtain images of living cells. Measurement of mechanical properties with these tips requires a contact model that takes into account their blunted geometry. The aim of this work was to develop a contact model of a blunted pyramidal tip and to assess the suitability of pyramidal tips for probing mechanical properties of soft gels and living cells. We developed a contact model of a blunted pyramidal tip indenting an elastic half-space. We measured Young's modulus (E) and the complex shear modulus (G*= G' +i G" ) of agarose gels and A549 alveolar epithelial cells with pyramidal tips and compared them with those obtained with spherical tips. The gels exhibited an elastic behavior with almost coincident loading and unloading force curves and negligible values of G". E fell sharply with indentation up to approximately 300 nm , showing a linear regime for deeper indentations. A similar indentation dependence of E with twofold lower values at the linear regime was obtained with the spherical tip fitted with Hertz's model. The dependence of E on indentation in cells paralleled that found in gels. Cells exhibited viscoelastic behavior with G"/G' approximately 1/4 . Pyramidal tips commonly used for AFM imaging are suitable for probing mechanical properties of soft gels and living cells. PMID:16196611

The structure of thin polymer blend films of polystyrene (PS) and poly-n-butyl-methacrylate (PnBMA) was examined with Transmission X-ray Microscopy (TXM), Scanning Force Microscopy (SFM), X-Ray Photoemission Electron Microscopy (X-PEEM) and Optical Microscopy (OM). Thin films were prepared by spin casting of a toluene solution of the polymer mixture onto silicon wafers retaining the native oxide. Depending on blend composition and annealing conditions smooth films with and without holes or films with well pronounced surface features (ribbons or islands) were produced. By TXM measurements a high lateral resolution study of the as cast and the annealed polymer blend samples was performed. The contrast in TXM is due to different absorption of x-radiation of the used polymers and due to variation in thickness. With X-PEEM the lateral distribution of the two polymers near the surface was mapped by employing the characteristic Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra of the polymers. The TXM technique is a microscopic method integrating over the total film thickness, whereas the X-PEEM technique is a highly surface sensitive method. TXM and X-PEEM are therefore complementary methods which provide important information on the structure of thin polymer blend films additional to the standard techniques SFM and OM.

The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown. PMID:26249564

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue. PMID:23442955

The goal of this work is to study the properties of living cells and cell membranes by using atomic force microscopy. During atomic force microscopy (AFM) measurement, there is a strong mechanical coupling between the AFM tip and the cell. The purpose of this paper is to present a model of the overall mechanical response of the cell that allows us to separate out the mechanical response of the cell from the local surface interactions we wish to quantify. These local interactions include recognition (or binding) events between molecules bound to an AFM tip (e.g., an antibody) and molecules or receptors on the cell surface (e.g., the respective antigen). The computational model differs from traditional Hertzian contact models by explicitly taking into account the mechanics of the biomembrane and cytoskeleton. The model also accounts for the mechanical response of the living cell during arbitrary deformation. The indentation of a bovine sperm cell is used to test the validity of this model, and further experiments are proposed to fully parameterize the model. PMID:11983924

The actuator functionality of electromechanical polypropylene films was studied using atomic force microscopy. The film carries a permanent electric charge and includes microbubbles as a result of two-dimensional stretching of the film. The thickness change of various film structures covered with electrodes was measured as a function of external voltage. The dependence was found to be nonlinear, the thickness change in the range 0.001%-0.1% of the total film thickness and affected by the internal charge density of the film. Applying a capacitor model including an air gap within the polymer layer enabled the determination of the Young's modulus, the interfacial charge density and the actuator sensitivity of the studied structures.

Conventional composite sol-gel method has been modified to enhance the piezoelectric performance of ceramic thick films. Lead zirconate titanate (PZT) and lead magnesium niobate-lead titanate (PMN-PT) thick films were fabricated using the modified sol-gel method for ultrasonic transducer applications. In this work, piezoresponse force microscopy was employed to evaluate the piezoelectric characteristics of PZT and PMN-PT composite sol-gel thick films. The images of the piezoelectric response and the strain-electric field hysteresis loop behavior were measured. The effective piezoelectric coefficient (d33,eff) of the films was determined from the measured loop data. It was found that the effective local piezoelectric coefficient of both PZT and PMN-PT composite films is comparable to that of their bulk ceramics. The promising results suggest that the modified composite sol-gel method is a promising way to prepare the high-quality, crack-free ceramic thick films. PMID:23798771

Atomic force microscopy (AFM) is one of many new technologies available to study the mechanical properties and mechanobiological responses of living cells. Despite the widespread usage of this technology, there has been little attempt to develop new theoretical frameworks to interpret the associated data. Rather, most analyses rely on the classical Hertz solution for the indentation of an elastic half-space within the context of linearized elasticity. In contrast, we propose a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the three primary structural constituents of the cytoskeleton. Moreover, we extend a prior solution for a small indentation superimposed on a finite equibiaxial extension by incorporating in this mixture model for the special case of an initially random distribution of constituents (actin, intermediate filaments, and microtubules). We submit that this theoretical framework will allow an improved interpretation of indentation force–depth data from a sub-class of atomic force microscopy tests and will serve as an important analytical check for future finite element models. The latter will be necessary to exploit further the capabilities of both atomic force microscopy and nonlinear mixture theories for cell behavior. PMID:15322929

Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

Background Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. Results We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. Conclusions Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses. PMID:24834016

Cells exhibit the ability to sense and respond to local mechanical stimuli, leading to changes in function. This capability, referred to as mechanotransduction, is essential to normal tissue function, but the exact mechanisms by which cells sense local forces (strain, shear, compression and vibration) remain unclear. Recent studies in small animals and humans indicate that the frequency of cyclic mechanical stimuli is important, with physiological responses observed for stimuli ranging between 1 and 90 Hz. To better understand the cellular and molecular mechanisms underlying mechanotransduction, it will be important to observe cells in real time, using optical microscopy during high-frequency mechanical stimulation. We have developed a motion-control platform that can produce sinusoidal vibration of live cells during simultaneous high-speed microscopy and fluorimetry, at frequencies up to 100 Hz with peak acceleration up to 9.8 m s-2. The platform is driven by a voice coil and acceleration is measured with an accelerometer (Dytran 7521A1). The motion waveform was verified by high-speed imaging, using a digital camera (Casio EX-F1) operating at 1200 frames s-1 attached to an inverted microscope (Nikon Diaphot). When operating at 45 Hz and 2.94 m s-2 peak acceleration, the observed motion waveform exhibited sinusoidal behaviour, with measured peak-to-peak amplitude of 72 μm. Cultured osteoblast-like cells (UMR-106) were subjected to 2.94 m s-2 vibration at 45 Hz and remained attached and viable. This device provides - for the first time - the capability to mechanically stimulate living cells while simultaneously observing responses with optical microscopy.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton. PMID:24022089

The localized charge and electrostatic properties of organic photovoltaic thin films are predominating factors for controlling energy conversion efficiency. The surface potential and electrostatic structures of organic photovoltaic thin films were investigated by frequency shift mode Kelvin force microscopy (KFM) and electrostatic force microscopy (EFM). The KFM images of a poly[2-methoxy-5-(3‧,7‧-dimethyloctyloxy)-1,4-phenylene vinylene]/phenyl-C61-butyric-acid-methyl ester (PCBM) blend thin film reveals that the PCBM domains precipitate as the topmost layer on the thin films. We find fine structures that were not observed in the topography and KFM images. The bias dependence of the EFM images suggests that the EFM contrast reflects the field-induced polarization, indicating the presence of charge trapping sites.

"Learn & Live," a documentary film created by The George Lucas Educational Foundation and hosted by actor Robin Williams, profiles four K-12 school programs that are seeing positive results. In addition to these stories, the film shares insights from experts in education and technology to help explain why the innovations profiled in the film are…

Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells' fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young's modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young's modulus induced by the action of a cytoskeleton-targeting drug.

Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells’ fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young’s modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young’s modulus induced by the action of a cytoskeleton-targeting drug.

High-temperature superconductors were discovered 20 years ago, inspiring dreams of levitating trains fed by superconducting power lines. The cuprates, particularly YBa2Cu3O7-delta (YBCO), still promise to fulfill such applications, but must be made to carry higher current density, Jc, which is limited by the rapid onset of dissipation. The dissipation arises from the movement of magnetic vortices in the material, driven by the magnetic field of the current. It is therefore natural to use magnetic imaging to understand these limits on the current. Initially, I fix a mesoscopic ring of YBCO to a micro-Hall sensor and demonstrate that the sensor is capable of detecting small numbers of vortices. I then proceed with magnetic imaging, constructing a cryogenic scanning Hall probe microscope that combines a 1 x 4 cm scan range with 200 nm positioning resolution by coupling stepper motors to high-resolution drivers and reducing gears. It enables me to image an entire sample, then zoom in on regions of interest, down to the level of an individual quantized vortex. Applying this capability to current-carrying YBCO strips, I generate magnetic movies of the materials' periodic response to applied ac currents. From the movies, I reconstruct current density by inverting the Biot-Savart law, and electric field by approximating dB/dt and using Faraday's law. I thereby obtain complete, space- and time-resolved characterizations of the materials, including maps of ac power losses. After demonstrating this analysis on a single-crystal film, I image two "coated conductors"---YBCO grown on metal tape. I find relatively homogeneous flux penetration in a film grown by pulsed laser deposition (PLD) on an ion beam assisted deposition (IBAD) substrate, which contrasts with the weak-link behavior of grain boundaries in a film grown by metalorganic deposition (MOD) on rolling assisted biaxially textured substrate (RABiTS). Nonetheless, the in-plane meandering of the MOD film's boundaries

In this paper we introduce mechanical and structural characteristics of diamond-like carbon (DLC) films which were prepared on silicon substrates by radio frequency (RF) plasma enhanced chemical vapor deposition (PECVD) method using methane (CH{sub 4}) and hydrogen (H{sub 2}) gas. The films were annealed at various temperatures ranging from 300 to 900 °C in steps of 200 °C using rapid thermal processor (RTP) in nitrogen ambient. Tribological properties of the DLC films were investigated by atomic force microscopy (AFM) in friction force microscopy (FFM) mode. The structural properties of the films were obtained by high resolution transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The wettability of the films was obtained using contact angle measurement. XPS analysis showed that the sp{sup 3} content is decreased from 75.2% to 24.1% while the sp{sup 2} content is increased from 24.8% to 75.9% when the temperature is changed from 300 to 900 °C. The contact angles of DLC films were higher than 70°. The FFM measurement results show that the highest friction coefficient value was achieved at 900 °C annealing temperature.

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium, cAMP, inositol phosphates and cGMP. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker. PMID

Ferroelectric lead zirconate titanate (PZT) thin films have been analysed using electron backscatter diffraction (EBSD). Grain orientation mapping has been demonstrated, showing that features smaller than 100 nm may be successfully indexed. In conjunction with piezoresponse force microscopy (PFM), which was used to map and quantify the piezoelectric response from the same region of the films with a resolution of 10 nm, an analysis of the effects of grain orientation on the measured response at the nanoscale was possible. The microtexture of the film showed the presence of both mono- and multi-domains within grains exhibiting sizes of hundreds of nanometres.

NMR is widely used in many fields due to its powerful advantages such as nondestructive, chemically selective detection, and local probing. However, because of its low sensitivity, it is difficult to investigate thin film samples by conventional NMR. MRFM is the combined technic of NMR and Scanning Probe Microscopy (SPM), and it enabled exceptional sensitivity increasement of NMR detection. We succeeded in detecting general thin film NMR spectrum for the first time by modifying the MRFM. CaF2 34nm thin film NMR was detected and we observed 20 Gauss spectrum in proximity to bulk spectrum which is about 10 Gauss.

In this letter, we report fluctuation microscopy studies of medium-range ordering in amorphous diamond-like carbon films and the effect of annealing on this ordering. Annealed and unannealed diamond-like carbon films have almost identical short-range order. Our fluctuation microscopy results, however, indicate the presence of medium range order or clustering in the films on a lateral length scale that exceeds 1 nm. Within the clustered regions, the dominant local ordering appears to be diamond-like, and graphite-like ordering is not observed. Thermal annealing up to 600 {sup o}C leads to an increase in diamond-like clustering with no onset of graphite-like clustering. However, after high temperature annealing up to 1000 {sup o}C, graphite-like clustering becomes apparent as a result of the conversion of diamond-like carbon to graphite-like carbon. The results on the as-deposited films and films annealed up to 600 {sup o}C suggest that a spontaneous medium range ordering process occurs in diamond-like carbon films during and subsequent to film growth, and this may play an important role in stress relaxation.

We propose a semi-automated pipeline for the detection of possible cell divisions in live-imaging microscopy and the classification of these mitosis candidates using a Convolutional Neural Network (CNN). We use time-lapse images of NIH3T3 scratch assay cultures, extract patches around bright candidate regions that then undergo segmentation and binarization, followed by a classification of the binary patches into either containing or not containing cell division. The classification is performed by training a Convolutional Neural Network on a specially constructed database. We show strong results of AUC = 0.91 and F-score = 0.89, competitive with state-of-the-art methods in this field. PMID:26736369

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range. PMID:25599551

Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events. PMID:24710546

Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range. PMID:25599551

In this work we present the coherence controlled holographic microscopy (CCHM)1 and its ability to image the living cells in turbid media2. The CCHM method and its advantages are introduced. A 'coherence gate effect'3, that enables imaging in turbid media, occurs owing to the low coherence illumination in our setup. The coherence gate effect is briefly theoretically explained and comparison of images with different illumination sources is shown. After that, the possibility of imaging in turbid media is applied to investigation of cell reactions to cytopathic turbid emulsions. In our experiments we used human cancer cells treated by biologically active phospholipids (BAPs). Cellular events leading to cell death, that would otherwise remain hidden in turbid media, are clearly observable and according to them cell fate can be deduced.

The nucleation and growth of plasma-enhanced chemical-vapor deposited polycrystalline diamond films were studied using atomic force microscopy (AFM). AFM images were obtained for (1) nucleated diamond films produced from depositions that were terminated during the initial stages of growth, (2) the silicon substrate-diamond film interface side of diamond films (1-4 micrometers thick) removed from the original surface of the substrate, and (3) the cross-sectional fracture surface of the film, including the Si/diamond interface. Pronounced tip effects were observed for early-stage diamond nucleation attributed to tip convolution in the AFM images. AFM images of the film's cross section and interface, however, were not highly affected by tip convolution, and the images indicate that the surface of the silicon substrate is initially covered by a small grained polycrystalline-like film and the formation of this precursor film is followed by nucleation of the diamond film on top of this layer. X-ray photoelectron spectroscopy spectra indicate that some silicon carbide is present in the precursor layer.

The nucleation and growth of plasma enhanced chemical vapor deposited (PECVD) polycrystalline diamond films were studied using atomic force microscopy (AFM). AFM images were obtained for: (1) nucleated diamond films produced from depositions that were terminated during the initial stages of growth, (2) the silicon substrate-diamond film interface side of diamond films (1-4 micrometers thick) removed from the original surface of the substrate, and (3) cross-sectional fracture surface of the film, including the Si/diamond interface. Pronounced tip effects were observed for early-stage diamond nucleation attributed to tip convolution in the AFM images. AFM images of the films cross-section and interface however were not affected by tip convolution, and the images indicate that the surface of the silicon substrate is initially covered by small grained polycrystalline-like film and the formation of this precursor film is followed by nucleation of the diamond film on top of this layer. X-ray photoelectron spectroscoy (XPS) spectra indicates that some silicon carbide is present in the precursor layer.

Protein-based nanofibrils are emerging as a promising class of materials that provide unique properties for applications such as biomedical and food engineering. Here, we use atomic force microscopy and stochastic optical reconstruction microscopy imaging to elucidate the growth dynamics, exchange kinetics, and polymerization mechanism for fibrils composed of a de novo designed recombinant triblock protein polymer. This macromolecule features a silk-inspired self-assembling central block composed of GAGAGAGH repeats, which are known to fold into a β roll with turns at each histidine and, once folded, to stack, forming a long, ribbon-like structure. We find several properties that allow the growth of patterned protein nanofibrils: the self-assembly takes place on only one side of the growing fibrils by the essentially irreversible addition of protein polymer subunits, and these fibril ends remain reactive indefinitely in the absence of monomer ("living ends"). Exploiting these characteristics, we can grow stable diblock protein nanofibrils by the sequential addition of differently labeled proteins. We establish control over the block length ratio by simply varying monomer feed conditions. Our results demonstrate the use of engineered protein polymers in creating precisely patterned protein nanofibrils and open perspectives for the hierarchical self-assembly of functional biomaterials. PMID:27124596

Spirochetes from microbial mats and anaerobic mud samples collected in salt marshes were studied by light microscopy, whole mount and thin section transmission electron microscopy. Enriched in cellobiose-rifampin medium, selective for Spirochaeta bajacaliforniensis, seven distinguishable spirochete morphotypes were observed. Their diameters ranged from 0.17 micron to > 0.45 micron. Six of these morphotypes came from southwest Cape Cod, Massachusetts: five from Microcoleus-dominated mat samples collected at Sippewissett salt marsh and one from anoxic mud collected at School Street salt marsh (on the east side of Eel Pond). The seventh morphotype was enriched from anoxic mud sampled from the north central Cape Cod, at the Sandy Neck salt marsh. Five of these morphotypes are similar or identical to previously described spirochetes (Leptospira, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirosymplokos deltaeiberi and Treponema), whereas the other two have unique features that suggest they have not been previously described. One of the morphotypes resembles Spirosymplokos deltaeiberi (the largest free-living spirochete described), in its large variable diameter (0.4-3.0 microns), cytoplasmic granules, and spherical (round) bodies with composite structure. This resemblance permits its tentative identification as a Sippewissett strain of Spirosymplokos deltaeiberi. Microbial mats samples collected in sterile Petri dishes and stored dry for more than four years yielded many organisms upon rewetting, including small unidentified spirochetes in at least 4 out of 100 enrichments.

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

Bleomycin has been used in the clinic as a chemotherapeutic agent for the treatment of several neoplasms, including non-Hodgkins lymphomas, squamous cell carcinomas, and testicular tumors. The effectiveness of bleomycin is believed to be derived from its ability to bind and oxidatively cleave DNA in the presence of a iron cofactor in vivo. A substantial amount of data on BLM has been collected, there is little information concerning the effects of bleomycin in living cells. In order to obtain data pertinent to the effects of BLM in intact cells, we have exploited the intrinsic fluorescence property of bleomycin to monitor the uptake of the drug in mammalian cells. We employed two light microscopy techniques, a wide-field and three-photon excitation (760 nm) fluorescence microscopy. Treatment of HeLa cells with bleomycin resulted in rapid to localization within the cells. In addition data collected from the wide field experiments, three-photon excitation of BLM which considerably reduced the phototoxic effect compared with UV light excitation in the wide-field microscopy indicated co-localization of the drug to regions of the cytoplasm occupied by the endoplasmic reticulum probe, DiOC5. The data clearly indicates that the cellular uptake of bleomycin after one minute includes the nucleus as well as in cytoplasm. Contrary to previous studies, which indicate chromosomal DNA as the target of bleomycin, the current findings suggest that the drug is distributed to many areas within the cell, including the endoplasmic reticulum, an organelle that is known to contain ribonucleic acids.

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

Demands of higher spatial and temporal resolutions in linear and nonlinear imaging keep pushing the limits of optical microscopy. We showed recently that a multiphoton microscope with 200 kHz repetition rate and wide-field illumination has a 2-3 orders of magnitude improved throughput compared to a high repetition rate confocal scanning microscope. Here, we examine the photodamage mechanisms and thresholds in live cell imaging for both systems. We first analyze theoretically the temperature increase in an aqueous solution resulting from illuminating with different repetition rates (keeping the deposited energy and irradiated volume constant). The analysis is complemented with photobleaching experiments of a phenolsulfonphthalein (phenol red) solution. Combining medium repetition rates and wide-field illumination promotes thermal diffusivity, which leads to lower photodamage and allows for higher peak intensities. A three day proliferation assay is also performed on living cells to confirm these results: dwell times can be increased by a factor of 3×10(6) while still preserving cell proliferation. By comparing the proliferation data with the endogenous two-photon fluorescence decay, we propose to use the percentage of the remaining endogenous two-photon fluorescence after exposure as a simple in-situ viability test. These findings enable the possibility of long-term imaging and reduced photodamage. PMID:27446668

Demands of higher spatial and temporal resolutions in linear and nonlinear imaging keep pushing the limits of optical microscopy. We showed recently that a multiphoton microscope with 200 kHz repetition rate and wide-field illumination has a 2–3 orders of magnitude improved throughput compared to a high repetition rate confocal scanning microscope. Here, we examine the photodamage mechanisms and thresholds in live cell imaging for both systems. We first analyze theoretically the temperature increase in an aqueous solution resulting from illuminating with different repetition rates (keeping the deposited energy and irradiated volume constant). The analysis is complemented with photobleaching experiments of a phenolsulfonphthalein (phenol red) solution. Combining medium repetition rates and wide-field illumination promotes thermal diffusivity, which leads to lower photodamage and allows for higher peak intensities. A three day proliferation assay is also performed on living cells to confirm these results: dwell times can be increased by a factor of 3×106 while still preserving cell proliferation. By comparing the proliferation data with the endogenous two-photon fluorescence decay, we propose to use the percentage of the remaining endogenous two-photon fluorescence after exposure as a simple in-situ viability test. These findings enable the possibility of long-term imaging and reduced photodamage.

Endogenous fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), keratin, and tryptophan, have been used as contrast agents for imaging metabolism and morphology of living cells and tissues. Multilabeling which maps the distribution of different targets is an indispensable technique in many biomedical and biochemical studies. Therefore, two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with in vivo fluorescence labeling techniques such as genetically encoded fluorescent protein could be a powerful tool for imaging living cells and tissues. However, the challenge is that the excitation and emission wavelengths of these endogenous fluorophores and fluorescence labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected Supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores and fluorescent proteins such as NADH, tryptophan, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) were excited in their optimal wavelengths alternately or simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and spectral domains. Cellular organelles such as nucleus, mitochondria, microtubule and Endoplasmic Reticulum (ER), were clearly revealed in the TPEF images.

This paper describes the investigation of elastical properties and imaging of living cochlear hair bundles of inner (IHC) and outer hair cells (OHC) on the level of individual stereocilia. A custom-made AFM-setup was used, allowing to scan the mechano-sensitive structures of the inner ear under direct control of an upright differential interference contrast (DIC) microscope with a water-immersion objective. Scanning electron microscopy (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that forces up to 1.5 nanonewton (nN) did not cause obvious damage of the surface morphology of the stereocilia. These are the first images of hair bundles of living sensory cells of the organ of Corti by AFM. They display the tips of individual stereocilia and the typical V-shape of ciliary bundles. Since line scans clearly show that slope and force interaction depend on the elastical properties of stereocilia, quantitative stiffness measurements and stimulation of single transduction channels are suggested. PMID:10741679

Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laser-scanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response. PMID:25383179

A lateral dislocation in a double-layer microsphere film is very difficult to identify because the constituent domains have the same two-dimensional crystalline orientation. Orientation-sensitive optical techniques cannot resolve this issue. Here, we demonstrate that partial dark-field (pDF) optical microscopy can be very effective in identifying this type of domain boundary and dislocation of a close-packed microsphere double-layer. Using the hexagonal symmetry of the close-packed microsphere film and the light-focusing property of microspheres, the partially blocked dark-field condenser can provide much higher contrast than other optical microscopy modes can in identifying the laterally dislocated domains. The former can also distinguish domains with different crystalline orientation by rotating the pDF stop. The simplicity of the pDF mode will make it an ideal tool for the structural study of close-packed double-layer microsphere films. PMID:25959375

A method for activation of high resistivity, (100) texture CVD diamond films with gold, to improve low field electron emission is described. A model based on the dielectric breakdown of the diamond film is proposed and a test experiment, which consists of heating the gold layer to a point where the gold forms sub-micron spheroids on the diamond surface, is described which supports the model. The deposition of carbon and sulfur on Mo(310) is characterized with scanning Auger Microscopy. Correlation between Photoelectron emission Microscopy, scanning Auger Microscopy and Auger spectroscopy can be made, so that individual features in PEEM and SAM images can be identified by elemental composition. The initial design of a Seeded Supersonic Molecular Beam system for diamond deposition is described.

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading. PMID:27063564

This PhD thesis presents the work on specific complex oxides and rare earth manganite thin films which were characterized mainly by transmission electron microscopy (TEM). The scientific results are divided in two main parts: the first part is devoted to the complex oxide films and the second to the rare earth manganite films. I. Complex oxides: The compositional influence of Cr, Al and Y on the microstructure of Mg-Cr-O, Mg-Al-O, Mg-Y-0 and Y-Al-O films synthesized by a reactive magnetron sputtering technique is reported. The study was based on a series of films with a range of compositions (metal ratios) deposited on Si substrates (without external substrate heating). The film thickness is about 1 μm (±200 nm). The effect of high temperatures (973 K to 1223 K) on the microstructural evolution of Mg-AlO, Mg-Cr-O and Y-Al-O films with specific metal ratios is also reported. II. Rare Earth Manganite Films: The microstructure and defect characterisation of hexagonal ReMnO3 (Re=Y, Tb, Dy, Ho and Er) thin films and multilayers is reported. The effect of off-stoichiometry on the microstructure of some hexagonal ReMnO3 (Re=Er, Dy and Ho) films with specific cationic ratios is also discussed. These thin films and multilayers were deposited on (111) YSZ and (111) Pt/TiO2/SiO 2/Si (stack) substrates by liquid injection metal organic chemical vapour deposition (MOCVD). The thickness of the films and multilayers is between 10 nm and 500 nm.

In this thesis we explore specific properties of the cytoskeleton and growth of living neurons via atomic force and fluorescence microscopies. We make the first comparative elastic modulus measurements on three types of neuronal cells plated on three types of substrate adhesion factors. We discover that during phases of active neurite extension the soma of cortical neurons stiffens reversibly due to changes in microtubule aggregation. Additionally, we demonstrate that mechanical properties of cortical neurons measured near physiological temperatures are primarily dependent on the microtubule component of the cytoskeleton. We further explore the response of the neuronal cytoskeleton to changes in ambient temperature. The elastic modulus of cortical neuron somas is discovered to increase dramatically upon a drop in ambient temperature. We determine through fluorescent staining and chemical modification of the cytoskeleton that this stiffening is due primarily to a change in the mechanically dominant component of the cytoskeleton from microtubules at 37ºC to actin at 25ºC precipitated by changes in myosin II dynamics within the cell. We make the first direct mechanical measurements of the pericellular brush layer on living neurons, demonstrating that the traditionally observed viscoelastic behavior of the neuronal soma is due to the properties of this brush layer. When the brush layer is excluded, the underlying soma is discovered to be both stiffer than previously observed, and elastic, with no loading-speed dependence to the elastic modulus under the test conditions. We additionally demonstrate that the soma elastic modulus, brush length, and brush density are all dependent on the ambient temperature. Finally, through fluorescent and bright field microscopies we track the outgrowth of living neurons on patterned directional surfaces, demonstrating that asymmetrical ratchet topographies unidirectionally bias axonal outgrowth. We model the outgrowth of the neurons

Metal phthalocyanines are attracting significant attention, owing to their potential for applications in chemical sensors, solar cells and organic magnets. As the electronic properties of molecular films are determined by their crystallinity and molecular packing, the optimization of film quality is important for improving the performance of organic devices. Here, we present the results of in situ low-energy electron microscopy / photoemission electron microscopy (LEEM/PEEM) studies of incorporation-limited growth of manganese-phthalocyanine (MnPc) on Ag(100) surfaces. MnPc thin films were grown on both, bulk Ag(100) surface and thin Ag(100)/Fe(100) films, where substrate spin-polarized electronic states can be modified through tuning the thickness of the Ag film. We also discuss the electronic structure and magnetic ordering in MnPc thin films, investigated by angle- and spin-resolved photoemission spectroscopy. Research carried out at the Center for Functional Nanomaterials and National Synchrotron Light Source, Brookhaven National Laboratory, which are supported by the U.S. Dept. of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.

One consistent limitation for high-resolution imaging of small nanoparticles is the high background signal from the amorphous carbon support film. With interest growing for smaller and smaller nanostructures, state of the art electron microscopes are becoming necessary for rudimentary tasks, such as nanoparticle sizing. As a monolayer of carbon, free-standing graphene represents the ultimate support film for nanoparticle imaging. In this work, conventional high-resolution transmission electron microscopy (HRTEM) and aberration-corrected scanning transmission electron microscopy (STEM) were used to assess the benefits and feasibility of few-layer graphene support films. Suspensions of few-layer graphene to produce the support films were prepared by simple sonication of exfoliated graphite. The greatest benefit was observed for conventional HRTEM, where lattice resolved imaging of sub 2 nm CdSe nanocrystals was achieved. The few-layer graphene films were also used as a support film for C(s)-corrected STEM and electron energy loss spectroscopy of CuInSe(2) nanocrystals. PMID:20356171

We have been developing a picosecond single shot soft x-ray contact microscopy system for observing the nanometer-scale inner structure of the living biological specimen in a hydrated condition. The microscopy system consists of an intense IR pump laser system for generating laser-induced plasma as a soft x-ray source and x-ray microscope chamber. The pump laser system employs OPCPA (Optical Parametric Chirped Pulse Amplification) technique to obtain a high contrast pump laser pulse, and we can generate water-window x-rays effectively by combining it to an ultra-thin metal target. The x-ray microscope chamber is composed of a vacuum chamber, a focusing lens, a metal film target, an in-vacuum type sample holder. The pump laser pulse is focused on the metal film target with a focusing lens. The soft x-rays from the laser-induced plasma illuminates bio-specimens on the PMMA photo resist set in the in-vacuum sample holder. The photo resist is developed and the x-ray transmission image recorded on the photo resist is read out by AFM. We took x-ray images of hydrated Leydig cells from mouse testicle and demonstrated that the developed x-ray microscopy system has a spatial resolution of about 100 nm.

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons. PMID:25735540

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons.

Direct observation of the film thickness, molecular structure and individual fluorine atoms from highly oriented poly(tetrafluoroethylene) (PTFE) films were achieved using atomic force microscopy (AFM). A thin PTFE film is mechanically deposited onto a smooth glass substrate at specific temperatures by a friction transfer technique. Atomic resolution images of these films show that the chain-like helical structures of the PTFE macromolecules are aligned parallel to each other with an intermolecular spacing of 5.72 A, and individual fluorine atoms are clearly observed along these twisted molecular chains with an interatomic spacing of 2.75 A. Furthermore, the first direct AFM measurements for the radius of the fluorine-helix, and of the carbon-helix in sub-angstrom scale are reported as 1.70 A and 0.54 A respectively.

Direct observation of the film thickness, molecular structure, and individual fluorine atoms from highly oriented poly(tetrafluoroethylene) (PTFE) films were achieved using atomic force microscopy (AFM). A thin PTFE film is mechanically deposited onto a smooth glass substrate at specific temperatures by a friction-transfer technique. Atomic resolution images of these films show that the chain-like helical structures of the PTFE macromolecules are aligned parallel to each other with an intermolecular spacing of 5.72 A, and individual fluorine atoms are clearly observed along these twisted molecular chains with an interatomic spacing of 2.75 A. Furthermore, the first direct AFM measurements for the radius of the fluorine-helix, and of the carbon-helix in sub-angstrom scale are reported as 1.7 and 0.54 A respectively.

Samples of chemically-vapor-deposited micrometer and sub-micrometer-thick films of polysilicon were analyzed by transmission electron microscopy (TEM) in cross-section and by Raman spectroscopy with illumination at their surface. TEM and Raman spectroscopy both find varying amounts of polycrystalline and amorphous silicon in the wafers. Raman spectra obtained using blue, green and red excitation wavelengths to vary the Raman sampling depth are compared with TEM cross-sections of these films. Films showing crystalline columnar structures in their TEM micrographs have Raman spectra with a band near 497 cm{sup {minus}1} in addition to the dominant polycrystalline silicon band (521 cm{sup {minus}1}). The TEM micrographs of these films have numerous faulted regions and fringes indicative of nanometer-scale silicon structures, which are believed to correspond to the 497cm{sup {minus}1} Raman band.

The indentation modulus of thin films of porous organosilicate glass with a nominal porosity content of 30% and thicknesses of 350nm, 200nm, and 46nm is determined with help of atomic force acoustic microscopy (AFAM). This scanning probe microscopy based technique provides the highest possible depth resolution. The values of the indentation modulus obtained for the 350nm and 200nm thin films were respectively 6.3GPa±0.2GPa and 7.2GPa±0.2GPa and free of the substrate influence. The sample with the thickness of 46nm was tested in four independent measurement sets. Cantilevers with two different tip radii of about 150nm and less than 50nm were applied in different force ranges to obtain a result for the indentation modulus that was free of the substrate influence. A detailed data analysis yielded value of 8.3GPa±0.4GPa for the thinnest film. The values of the indentation modulus obtained for the thin films of porous organosilicate glasses increased with the decreasing film thickness. The stiffening observed for the porous films could be explained by evolution of the pore topology as a function of the film thickness. To ensure that our results were free of the substrate influence, we analyzed the ratio of the sample deformation as well as the tip radius to the film thickness. The results obtained for the substrate parameter were compared for all the measurement series and showed, which ones could be declared as free of the substrate influence. PMID:26799327

Solid state dewetting and the subsequent morphological changes for platinum thin films grown on zinc oxide (ZnO) buffered (001) silicon substrates (Pt/ZnO/SiO{sub 2}/(001)Si system) is investigated under vacuum conditions via a custom-designed confocal laser microscope coupled with a laser heating system. Live imaging of thin film dewetting under a range of heating and quenching vacuum ambients reveals events including hillock formation, hole formation, and hole growth that lead to formation of a network of Pt ligaments, break up of Pt ligaments to individual islands and subsequent Pt islands shape reformation, in chronological fashion. These findings are corroborated by ex-situ materials characterization and quantitative electron microscopy analysis. A secondary hole formation via blistering before film rupture is revealed to be the critical stage, after which a rapid dewetting catastrophe occurs. This process is instantaneous and cannot be captured by ex-situ methods. Finally, an intermetallic phase forms at 900 °C and alters the morphology of Pt islands, suggesting a practical limit to the thermal environments that may be used for these platinized silicon wafers in vacuum conditions.

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5–25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument’s light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells. PMID:26200860

We introduce near-infrared Coherent Anti-Stokes Raman Scattering (CARS) microscopy as a method for the monitoring of fat deposition in a living organism by directly probing the CH II vibration of the lipids without the need for staining or labeling. This study nicely brings forward all the advantages of the technique: deep probe depth, low excitation powers, high 3-dimensional resolution, and visualization without the interference of exogenous label molecules, or fixation and staining procedures. Differences in fat deposition during the life cycle of the nematode Caenorhabditis elegans were evaluated quantitatively from the CARS microscopy images, showing that the technique can be used to study mechanisms that regulate lipid storage. Beside the wild type nematode, the feeding-deficient mutant pha-3 was studied. It was shown that the embryonal accumulation of energy stores is enough for the development of a full-sized pre-adult larva, being possible also for the mutant. However, the volume density of lipid stores at the fourth and last pre-adult development stage seems to determine its adult body size. Whereas the wild type larva maintains its size when becoming adult, though at the cost of reduced lipid density, the feeding deficient mutant instead has to reduce its body size in order to reach the same volume density of lipid stores. Both strains start off their adult life with a volume fraction of lipid stores corresponding to 6-7%; the wild type with a radius of 24+/-2 Âµm and the pha-3 mutant with a significantly smaller radius of 16+/-3 μm.

We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5–100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5–3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences. PMID:22611191

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells. PMID:26200860

Introduction Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. Method Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. Results The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties. Conclusion We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented. PMID:22242152

Photoillumination of mixed films comprised of the photopolymerizable fatty acid 10,12 pentacosadiynoic acid and perfluorotetradecanoic acid deposited onto glass substrates gives rise to the formation of oriented polydiacetylene photopolymer fibers. The degree of polymer fiber orientation was investigated using dual-view, polarized fluorescence microscopy of the polydiacetylene, which allowed for characterization of individual fluorescent polymer fibers after photopolymerization, as well as comparison of the orientation of different fibers within the same sample. Measurements indicated that individual fibers consisted of multiple photopolymer strands with various orientations, and that there was a preferred orientation for fibers in the film as a whole. The fibers were preferentially oriented at an angle of approximately 60° to the direction of film compression during deposition from a Langmuir trough, with orientation being the result of mechanical stress exerted by the compression barriers coupled with rotation of the polymer fibers during film draining. These measurements were complemented with conventional “bulk” fluorescence polarization experiments, and compared with mixed film structures described previously for these systems at the air-water interface using Brewster angle microscopy.

Photoillumination of mixed films comprised of the photopolymerizable fatty acid 10,12 pentacosadiynoic acid and perfluorotetradecanoic acid deposited onto glass substrates gives rise to the formation of oriented polydiacetylene photopolymer fibers. The degree of polymer fiber orientation was investigated using dual-view, polarized fluorescence microscopy of the polydiacetylene, which allowed for characterization of individual fluorescent polymer fibers after photopolymerization, as well as comparison of the orientation of different fibers within the same sample. Measurements indicated that individual fibers consisted of multiple photopolymer strands with various orientations, and that there was a preferred orientation for fibers in the film as a whole. The fibers were preferentially oriented at an angle of approximately 60° to the direction of film compression during deposition from a Langmuir trough, with orientation being the result of mechanical stress exerted by the compression barriers coupled with rotation of the polymer fibers during film draining. These measurements were complemented with conventional "bulk" fluorescence polarization experiments, and compared with mixed film structures described previously for these systems at the air-water interface using Brewster angle microscopy. PMID:24747858

The bonding, chemistry and ordering of molecular adsorbates on well defined single crystal surfaces and in ultrathin films was to be studied in an effort to develop sufficient fundamental understanding to allow the controlled preparation of anisotropic ultrathin films of organic monolayers. In this research the authors combine the use of optical probes (Raman spectroscopy, laser induced thermal desorption with Fourier transform mass spectrometry detection) with scanning tunneling microscopy (STM) and conventional methods of UHV surface science (Auger electron spectroscopy, x-ray photoelectron spectroscopy, low energy electron diffraction, and thermal desorption spectroscopy). The conventional surface probes provide well tested methods for the preparation and characterization of single crystal substrates. The optical probes used in the experiments provide powerful methods for the molecular identification of adsorbates in monolayers and ultrathin films. Scanning tunneling microscopy provides one with the ability to determine the detailed molecular level ordering of the molecular adsorbates. The emphasis of this research is on more complex molecular absorbates some of which are monomer precursors to ultrathin polymer films. Enhanced methods of Raman spectroscopy have been developed for the study of monolayer adsorbates on surfaces in ultrahigh vacuum environments. This report gives an overview of recent research results, including the construction of UHV variable temperature STM, analysis of STM images, growth and chemistry of intermetallic single crystal ultrathin films, and electron beam induced chemistry of tetracyanoquinodimethane.

Conjugated polymers are highly tunable organic semiconductors, which can be solution processed to form thin films, making them prime candidates for organic photovoltaic devices. One of the most important parameters in a conjugated polymer solar cell is the exciton diffusion length, which depends on intermolecular couplings, and is typically on the order of 10 nm. This mean exciton migration can vary dramatically between films and within a single film due to heterogeneities in morphology on length scales of 10's to 100's nm. To study the variability of exciton diffusion and morphology within individual conjugated polymer films, we are adapting stimulated emission depletion microscopy. STED is typically used in biology with well-engineered fluorescent labels or on NV-centers in diamond. I will, however, describe how we have demonstrated STED in conjugated polymer films of MEH-PPV and CN-PPV by taking care to first understand the film's photophysical properties. This new approach provides a way to study exciton diffusion by utilizing subdiffraction optical excitation volumes. In this way, we will obtain a spatiotemporal map of exciton distributions that will help to correlate the energetic landscape to film morphology at the nanoscale. This research is supported in part by the Department of Energy Office of Science Graduate Fellowship Program (DOE SCGF), made possible in part by the American Recovery and Reinvestment Act of 2009, administered by ORISE-ORAU under Contract No. DE-AC05-06.

Back-etch methods have been widely used to prepare plan view transmission electron microscopy (TEM) samples of thin films on membranes by removal of the Si substrate below the membrane by backside etching. The conventional means to determine when to stop the etch process is to observe the color of the light transmitted through the sample, which is sensitive to the remaining Si thickness. However, most metallic films thicker than 75 nm are opaque, and there is no detectable color change prior to film perforation. In this paper, a back-etch method based on the observation of an abrupt change of optical reflection contrast is introduced as a means to determine the etch endpoint to prepare TEM samples for these films. As the acid etchant removes the Si substrate material a rough interface is generated. This interface becomes a relatively smooth and featureless region when the etchant reaches the membrane (film/SiO2). This featureless region is caused by the mirror reflection of the film plane (film/SiO2 interface) through the optically transparent SiO2 layer. The lower etch rate of SiO2 (compared with Si) gives the operator enough time to stop the etching without perforating the film. A clear view of the morphology and control of Si roughness during etching are critical to this method, which are discussed in detail. The procedures of mounting wax removal and sample rinsing are also described in detail, as during these steps damage to the membrane may easily occur without appropriate consideration. As examples, the preparation of 100-nm-thick Fe-based amorphous alloy thin film and 160-nm-thick Cu-thin film samples for TEM imaging is described. PMID:18227137

Polythiophene nanoparticles were synthesized by iron catalyzed oxidative polymerization method. Polythiophene formation was detected by UV-Visible spectroscopy with λmax 375nm. Thin films of CTAB stabilized polythiophene nanoparticles was deposited on n-type silicon wafer by spin coating technique at 3000rpm in three cycles. Thickness of the thin films was computed as 300-350nm by ellipsometry. Atomic force micrscopyrevealws the particle size of polymeric nanoparticles in the range of 30nm to 100nm. Roughness of thinfilm was also analyzed from the atomic force microscopy data by Picoimage software. The observed RMS value lies in the range of 6 nm to 12 nm.

Nanosecond scale threshold switching is investigated with conducting atomic force microscopy (AFM) for an amorphous GeSe film. Switched bits exhibit 2–3 orders of magnitude variations in conductivity, as demonstrated in phase change based memory devices. Through the nm-scale AFM probe, this crystallization was achieved with pulse durations of as low as 15 ns, the fastest reported with scanning probe based methods. Conductance AFM imaging of the switched bits further reveals correlations between the switched volume, pulse amplitude, and pulse duration. The influence of film heterogeneities on switching is also directly detected, which is of tremendous importance for optimal device performance.

The thermal properties of amorphous and crystalline phases in chalcogenide phase change materials (PCM) play a key role in device performance for non-volatile random-access memory. Here, we report the nanothermal morphology of amorphous and crystalline phases in laser pulsed GeTe and Ge2Sb2Te5 thin films by scanning thermal microscopy (SThM). By SThM measurements and quantitative finite element analysis simulations of two film thicknesses, the PCM thermal conductivities and thermal boundary conductances between the PCM and SThM probe are independently estimated for the amorphous and crystalline phase of each stoichiometry.

Electron holography is applied for the study of the leakage field of thin-film ferromagnetic tips used as probes in magnetic force microscopy. We used commercially available pyramidal tips covered on one face with a thin NiCo film, which were then placed in a high external magnetic field directed along the pyramid axis. Good agreement between simulated and experimental electron phase difference maps allows to measure the local flux from the ferromagnetic tips and therefore to evaluate the perturbation induced by the microprobe stray field on the sample area. {copyright} {ital 1996 American Institute of Physics.}

Electrostatic Force Microscopy measurements have been performed on thin dielectric films on conducting substrates. Cantilever oscillation amplitude versus distance curves are measured as a function of the frequency of the voltage applied between tip and sample. When a DC voltage is applied, the oscillation amplitude versus distance curve is significantly different from that when a low frequency (500 Hz) AC voltage is applied (cantilever resonance at 125 kHz). The frequency dependence of the AC force response for different dielectric films (SiO2 and Al_2O_3) are studied. The frequency dependence is accounted for by the movement of charge near the sample surface with a finite response time.

Detailed studies of the growth and structure of thin films of metal oxides grown on metal single crystal surfaces using Scanning Tunneling Microscopy (STM) are presented. The oxide overlayer systems studied are iron oxide and titanium oxide on the Pt(III) surface. The complexity of the metal oxides and large lattice mismatches often lead to surface structures with large unit cells. These are particularly suited to a local real space technique such as scanning tunneling microscopy. In particular, the symmetry that is directly observed with the STM elucidates the relationship of the oxide overlayers to the substrate as well as distinguishing, the structures of different oxides.

During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs. PMID:24103784

Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.

Sodium ions (Na(+)) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na(+) concentrations ([Na(+)]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na(+)-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na(+)-sensitivity appropriate for recordings in living cells. The Na(+)-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na(+)]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na(+)]i rise in cockroach salivary gland cells, which was dependent on a Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems. PMID:25311309

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.

Atomic force microscopy (AFM) is a known method to study various surfaces. Here we report on the use of AFM to study surface oscillations (coming from the work of internal organs) of living organisms, like insects. As an example, ladybird beetles (Hippodamia convergens) measured in different parts of the insect at picometer level. This allows us to record a much broader spectral range of possible surface vibrations (up to several kHz) than the previously studied oscillations due to breathing, heartbeat cycles, coelopulses, etc. (up to 5 -10 Hz). The used here AFM method allows collecting signal from the area as small as ˜100nm2 (0.0001μm2) with an example of noise level of (2±0.2)x10-3 nm r.m.s. at the range of frequencies >50Hz (potentially, up to a MHz). Application of this method to humans is discussed. The method, being a relatively non-invasive technique providing a new type of information, may be useful in developing of what could be called ``nanophysiology.''

Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

Summary Engineered nanomaterials are known to enter human cells, often via active endocytosis. Mechanistic details of the interactions between nanoparticles (NPs) with cells are still not well enough understood. NP size is a key parameter that controls the endocytic mechanism and affects the cellular uptake yield. Therefore, we have systematically analyzed the cellular uptake of fluorescent NPs in the size range of 3.3–100 nm (diameter) by live cells. By using spinning disk confocal microscopy in combination with quantitative image analysis, we studied the time courses of NP association with the cell membrane and subsequent internalization. NPs with diameters of less than 10 nm were observed to accumulate at the plasma membrane before being internalized by the cells. In contrast, larger NPs (100 nm) were directly internalized without prior accumulation at the plasma membrane, regardless of their surface charges. We attribute this distinct size dependence to the requirement of a sufficiently strong local interaction of the NPs with the endocytic machinery in order to trigger the subsequent internalization. PMID:25551067

Mitochondria have a central role in cellular (patho)physiology, and they display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol that allows unbiased and automated quantification of mitochondrial 'morphofunction' (i.e., morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living primary human skin fibroblasts (PHSFs). Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester (TMRM), calcein-AM (acetoxy-methyl ester) and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data are subjected to a quality control (QC) algorithm based upon principal component analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. The protocol requires a time investment of ∼4 h distributed over 2 d. Although it is specifically developed for PHSFs, which are widely used in preclinical research, the protocol is portable to other cell types and can be scaled up for implementation in high-content screening. PMID:27560174

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50 nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed. PMID:26133539

Composite Ti/hydrocarbon plasma polymer films with different Ti concentration were deposited on silicon by dc magnetron sputtering of titanium in an atmosphere of argon and hexane. As measured by Kelvin force microscopy and visco-elastic atomic force microscopy, respectively, surface potential and hardness increase with increasing Ti content. Adhesion force to silicon and to fibrinogen molecules was stronger for the Ti-rich films as evaluated from the AFM force-distance curves. Fibrinogen forms a very soft layer on these composites with part of the protein molecules embedded in the outermost region of the plasma polymer. An increase of the surface charge due to fibrinogen adsorption has been observed and attributed to positively charged αC domains of fibrinogen molecule.

Azo copolymers are nonlinear-optical materials, in which polar orientation can be induced by optical poling or electrical poling. We report a new efficient approach to performing photo-assisted poling (PAP) by atomic force microscopy (AFM) for azo copolymer films containing disperse-red-1 chromophores, and to characterize the polar orientation by electrostatic force microscopy (EFM) at the submicrometre scale. Both PAP and contact electrification effects can be generated by the physical interaction between the probes and the films. We demonstrated that these two effects can be distinguished by the relationship between the signs of the charges (bound charges and transferred charges) and the probe bias. Finally, we achieve local PAP far below the glass transition temperature by AFM operated in the tapping mode, and the response of the polar chromophores to local PAP can be studied by EFM.

Low-energy electron microscopy is employed to image defects at buried interfaces through the strains they cause at the front surface. The interfacial defects studied here occur in high quality films of Mo(110) grown by molecular beam epitaxy on Al2O3(112¯0). The defects include steps and inclusions on the original sapphire surface and interfacial dislocations created where epitaxial strain causes slip.

As a tribute to the scientific work of Professor Gareth Thomas in the field of structure-property relationships this paper delineates a new possibility of Lorentz transmission electron microscopy (LTEM) to study the magnetic properties of soft magnetic films. We show that in contrast to the traditional point of view, not only does the direction of the magnetization vector in nano-crystalline films make a correlated small-angle wiggling, but also the magnitude of the magnetization modulus fluctuates. This fluctuation produces a rapid modulation in the LTEM image. A novel analysis of the ripple structure in nano-crystalline Fe-Zr-N film corresponds to an amplitude of the transversal component of the magnetization deltaMy of 23 mT and a longitudinal fluctuation of the magnetization of the order of deltaMx = 30 mT. The nano-crystalline (Fe99Zr1)1-xNx films have been prepared by DC magnetron reactive sputtering with a thickness between 50 and 1000 nm. The grain size decreased monotonically with N content from typically 100 nm in the case of N-free films to less than 10 nm for films containing 8 at%. The specimens were examined with a JEOL 2010F 200 kV transmission electron microscope equipped with a post column energy filter (GIF 2000 Gatan Imaging Filter). For holography, the microscope is mounted with a biprism (JEOL biprism with a 0.6 microm diameter platinum wire). PMID:12533225

A bimodal magnetic force microscopy (MFM) that uses an external magnetic field for the detection and imaging of magnetic thin films is developed. By applying the external modulation magnetic field, the vibration of a cantilever probe is excited by its magnetic tip at its higher eigenmode. Using magnetic nanoparticle samples, the capacity of the technique which allows single-pass imaging of topography and magnetic forces is demonstrated. For the detection of magnetic properties of thin film materials, its signal-to-noise ratio and sensitivity are demonstrated to be superior to conventional MFM in lift mode. The secondary resonance MFM technique provides a promising tool for the characterization of nanoscale magnetic properties of various materials, especially of magnetic thin films with weak magnetism.

The depth characterization of photopolymer films by confocal Raman microscopy is often troublesome due to refraction effects. To minimize these effects, we used an oil immersion objective and a method was developed to avoid penetration of the oil without damaging the sample surface. Since the surface may be sticky if oxygen in the air inhibits the photopolymerization, a protective layer could not be put onto the film. Therefore, the method consists in using a thin polypropylene foil as substrate for the coating and placing the sample upside down under the objective. In this manner, the immersion oil could be deposited on top of the polypropylene. The advantage of this setup is that the oil, polypropylene substrate, and photopolymer film have close refractive indices. Basic calculations showed that the depth resolution is hardly affected in that configuration and double-bond conversion profiles could be plotted as a function of reliable nominal depth. The validity of the methodology was confirmed by experiments carried out with a dry metallurgical objective on the sample surface, face up, where refraction effects are still minor. In addition, infrared spectroscopy, which was used to follow the photopolymerization, corroborated the Raman conversion of the films over their thickness. The confocal Raman microscopy method can be applied to various photopolymerized systems to characterize their behavior towards oxygen inhibition and other heterogeneities in conversion arising from inner filter effects or interactions between additives for instance. PMID:21986072

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can

Cellular electrets polymer is a new ferroelectret material exhibiting large piezoelectricity and has attracted considerable attentions in researches and industries. Property characterization is very important for this material and current investigations are mostly on macroscopic properties. In this work, we conduct nanoscale piezoelectric and ferroelectric characterizations of cellular polypropylene (PP) films using piezoresponse force microscopy (PFM). First, both the single-frequency PFM and dual-frequency resonance-tracking PFM testings were conducted on the cellular PP film. The localized piezoelectric constant d{sub 33} is estimated to be 7–11pC/N by correcting the resonance magnification with quality factor and it is about one order lower than the macroscopic value. Next, using the switching spectroscopy PFM (SS-PFM), we studied polarization switching behavior of the cellular PP films. Results show that it exhibits the typical ferroelectric-like phase hysteresis loops and butterfly-shaped amplitude loops, which is similar to that of a poly(vinylidene fluoride) (PVDF) ferroelectric polymer film. However, both the phase and amplitude loops of the PP film are intensively asymmetric, which is thought to be caused by the nonzero remnant polarization after poling. Then, the D-E hysteresis loops of both the cellular PP film and PVDF film were measured by using the same wave form as that used in the SS-PFM, and the results show significant differences. Finally, we suggest that the ferroelectric-like behavior of cellular electrets films should be distinguished from that of typical ferroelectrics, both macroscopically and microscopically.

The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the

We formulate the thin-film hydrodynamics of a suspension of polar self-driven particles and show that it is prone to several instabilities through the interplay of activity, polarity and the existence of a free surface. Our approach extends, to self-propelling systems, the work of Ben Amar and Cummings [Phys Fluids 13 (2001) 1160] on thin-film nematics. Based on our estimates the instabilities should be seen in bacterial suspensions and the lamellipodium, and are potentially relevant to the morphology of biofilms. We suggest several experimental tests of our theory.

We formulate the thin-film hydrodynamics of a suspension of polar self-driven particles and show that it is prone to several instabilities through the interplay of activity, polarity, and the existence of a free surface. Our approach extends, to self-propelling systems, the work of Ben Amar and Cummings [Phys. Fluids 13 1160 (2001)10.1063/1.1359748] on thin-film nematics. Based on our estimates the instabilities should be seen in bacterial suspensions and the lamellipodium, and are potentially relevant to the morphology of biofilms. We suggest several experimental tests of our theory. PMID:19392245

We formulate the thin-film hydrodynamics of a suspension of polar self-driven particles and show that it is prone to several instabilities through the interplay of activity, polarity, and the existence of a free surface. Our approach extends, to self-propelling systems, the work of Ben Amar and Cummings [Phys. FluidsPHFLE61070-6631 13 1160 (2001)10.1063/1.1359748] on thin-film nematics. Based on our estimates the instabilities should be seen in bacterial suspensions and the lamellipodium, and are potentially relevant to the morphology of biofilms. We suggest several experimental tests of our theory.

We present a photoacoustic microscopy (PAM) system based on a Fabry-Perot Interferometer (FPI) consisting of a transparent Polydimethylsiloxane (PDMS) thin film. Most of the PAM systems have limitations with the system alignment because the ultrasound transducers for detection are not transparent. Therefore, the excitation laser source should avoid the opaque transducer to illuminate the sample, which makes the system difficult to build-up. Especially, the system volume is highly limited to be compact. In our experiment, to solve these difficulties, a FPI based on the PDMS film has been implemented and applied to measure the acoustic wave signal. The system uses a FPI as an acoustic wave detector instead of a conventional ultrasound transducer. A tunable laser was used to choose the quadrature-point at which the signal has the highly sensitve and linear response to the acoustic wave. Also a 20Hz pulsed Nd:YAG laser was used to generate acoustic waves from a sample. When the acoustic waves arrive at the PDMS film, one of the surfaces of the film is modulated at the detecting point, which gives the tuned FPI interference signal. From the signal arriving time, the depth location of the sample is calculated. As a primary experiment using the PDMS thin film as an ultrasound transducer, a couple of narrow black friction tapes located in a water container were used as the samples. This proposed imaging method can be used in various applications for the detection and measurement of acoustic waves.

Soft x-ray spectromicroscopy has been used to investigate the degree of the molecular alignment of sulfonated benzo[de]benzo[4.5]imidazo[2,1-a]isoquinoline[7,1], a lyotropic chromonic liquid crystal (LCLC). LCLC thin films cast from concentrated aqua solution (20%wt.) , aligned by shear flow and dried, show strong linear dichroism in their C-, N-, O-, S- K edge near edge x-ray spectra (NEXAFS). The carbon K edge has been used for quantitative evaluation of the orientational texture of the films at a submicron spatial scale. This has verified there is predominantly in-plane alignment of the LC director. To highlight the role of hydrophobic-hydrophilic interactions, two stereoisomers of the same dye has been synthesized with different positioning of terminal sulfonate groups, in the form of a mixture of isomers with sulfonate groups in 2,10 and 2,11 positions (Y104 compound) and in a 5,10-disulfo arrangement (Y105). Both compounds develop characteristic herringbone-type texture with similar domain sizes. Polarized optical microscopy and higher resolution x-ray microscopy show sinusoidal-like undulations of the molecular director, with occasional crisscross appearance. Such behavior is found to be consistent with earlier observation of striations, characteristic of the columnar phase. The drastic difference in the degree of undulation ( ±15° in Y104 and ±7° in Y105 films) and long period of undulation (approaching the film thickness) requires further analysis. It was also found that the degree of in-plane order within domains changes from 0.8 for Y104 to >0.9 in Y105 films.

Soft x-ray spectromicroscopy has been used to investigate the degree of the molecular alignment of sulfonated benzo[de]benzo[4.5]imidazo[2,1-a]isoquinoline[7,1], a lyotropic chromonic liquid crystal (LCLC). LCLC thin films cast from concentrated aqua solution (20%wt.) , aligned by shear flow and dried, show strong linear dichroism in their C-, N-, O-, S- K edge near edge x-ray spectra (NEXAFS). The carbon K edge has been used for quantitative evaluation of the orientational texture of the films at a submicron spatial scale. This has verified there is predominantly in-plane alignment of the LC director. To highlight the role of hydrophobic-hydrophilic interactions, two stereoisomers of the same dye has been synthesized with different positioning of terminal sulfonate groups, in the form of a mixture of isomers with sulfonate groups in 2,10 and 2,11 positions (Y104 compound) and in a 5,10-disulfo arrangement (Y105). Both compounds develop characteristic herringbone-type texture with similar domain sizes. Polarized optical microscopy and higher resolution x-ray microscopy show sinusoidal-like undulations of the molecular director, with occasional crisscross appearance. Such behavior is found to be consistent with earlier observation of striations, characteristic of the columnar phase. The drastic difference in the degree of undulation ( +/-15 degrees in Y104 and +/-7 degrees in Y105 films) and long period of undulation (approaching the film thickness) requires further analysis. It was also found that the degree of in-plane order within domains changes from 0.8 for Y104 to >0.9 in Y105 films. PMID:18233857

In a recent work, we showed that atomic force microscopy (AFM) is a powerful technique to image cross sections of polycrystalline thin films. In this work, we apply a modification of AFM, namely, electrostatic force microscopy (EFM), to investigate the electronic properties of cleaved II-VI and multijunction thin-film solar cells. We cleave the devices in such a way that they are still working with their nominal photovoltaic efficiencies and can be polarized for the measurements. This allows us to differentiate between surface effects (work function and surface band bending) and bulk device properties. In the case of polycrystalline CdTe/CdS/SnO2/glass solar cells, we find a drop of the EFM signal in the area of the CdTe/CdS interface (±50 nm). This drop varies in amplitude and sign according to the applied external bias and is compatible with an n-CdS/p-CdTe heterojunction model, thereby invalidating the possibility of a deeply buried n-p CdTe homojunction. In the case of a triple-junction GaInP/GaAs/Ge device, we observe a variation of the EFM signal linked to both the material work-function differences and to the voltage bias applied to the cell. We attempt a qualitative explanation of the results and discuss the implications and difficulties of the EFM technique for the study of such thin-film devices.

Three-dimensional confocal laser scanning microscopy (CLSM) and two-photon excitation microscopy (TPEM) were used to study the response of cellular systems to fuzzy organized nanostructured polyelectrolytes used both as microcontainers and microcarriers for drug delivery. These nanostructured systems are named Nanocapsules and represent a new class of controllable colloids. CLSM and TPEM uniquely allow to follow the fate of encapsulated living cells and to track the pathway of nanocapsules introduced into cellular systems. For the former situation, it will be shown how living cells can be encapsulated and demonstrated the preservation of the metabolic and duplicating activity. In this case the role of the Nanocapsule is as microcontainer endowed of functionalized surface and of protective ability. The latter situation, is related to feeding living cells with Nanocapsules. This experiment serves in elucidating the comprehension of the potential cytotoxicity and of the ability of Nanocapsules to reach specific targets where active compounds can be released. Cellular systems used within this research are Saccharomyces cerevisiae and Paramecium primaurelia living cells. In the case of encapsulation of Saccharomyces cerevisiae living cells, the most relevant result is that, after encapsulation, cells preserve their metabolic activities and they are still able to divide. At this stage is also relevant the utilization of spectroscopic methods like fluorescence lifetime and second harmonic imaging. These hybrid polyelectrolyte-cells can provide a cheap model system in a wide range of biophysical and biotechnological applications, thanks to the tunable properties of the polyelectrolyte shell.

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines. PMID:26114028

Atomic force microscopy (AFM) is difficult to achieve in living mammals but is necessary for understanding mechanical properties of tissues in their native form in organisms. Here we report in vivo nanomechanical imaging of blood-vessel tissues directly in living mammalians by AFM combined with surgical operations. Nanomechanical heterogeneity of blood vessels is observed across the diverse microenvironments of the same tissues in vivo. This method is further used to measure the counteractive nanomechanical changes in real time during drug-induced vasodilation and vasoconstriction in vivo, demonstrating appealing potential in characterization of in vivo nanomechanical dynamics of native tissues.

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies (1-3). These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres (4-8). Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell. Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest (9). The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed. This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to

FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells.

We have grown Li-doped ZnO films on silicon (100) using the rf planar magnetron sputtering method. The surface charges induced piezoelectrically by defect and by polarization can be observed by electric force microscopy. The Li-doped ZnO films have been proven to be ferroelectric. The Raman spectra of ZnO and Li-doped ZnO films have been measured.

Sexual reproduction ensures propagation of species and enhances genetic diversity within populations. In flowering plants, sexual reproduction requires complicated and multi-step cell-to-cell communications among male and female cells. However, the confined nature of plant reproduction processes, which occur in the female reproductive organs and several cell layers of the pistil, limits our ability to observe these events in vivo. In this review, we discuss recent live-cell imaging in in vitro systems and the optical manipulation techniques that are used to capture the dynamic mechanisms representing molecular and cellular communications in sexual plant reproduction. PMID:23438900

A temperature dependence characterization system of microwave permeability of magnetic thin film up to 5 GHz in the temperature range from room temperature up to 423 K is designed and fabricated as a prototype measurement fixture. It is based on the near field microwave microscopy technique (NFMM). The scaling coefficient of the fixture can be determined by (i) calibrating the NFMM with a standard sample whose permeability is known; (ii) by calibrating the NFMM with an established dynamic permeability measurement technique such as shorted microstrip transmission line perturbation method; (iii) adjusting the real part of the complex permeability at low frequency to fit the value of initial permeability. The algorithms for calculating the complex permeability of magnetic thin films are analyzed. A 100 nm thick FeTaN thin film deposited on Si substrate by sputtering method is characterized using the fixture. The room temperature permeability results of the FeTaN film agree well with results obtained from the established short-circuited microstrip perturbation method. Temperature dependence permeability results fit well with the Landau-Lifshitz-Gilbert equation. The temperature dependence of the static magnetic anisotropy H(K)(sta), the dynamic magnetic anisotropy H(K)(dyn), the rotational anisotropy H(rot), together with the effective damping coefficient α(eff), ferromagnetic resonance f(FMR), and frequency linewidth Δf of the thin film are investigated. These temperature dependent magnetic properties of the magnetic thin film are important to the high frequency applications of magnetic devices at high temperatures. PMID:21895260

Conjugated polymers are highly tunable organic semiconductors, which can be solution processed to form thin films, making them prime candidates for organic photovoltaic devices. One of the most important parameters in a conjugated polymer solar cell is the exciton diffusion length, which depends on intermolecular couplings, and is typically on the order of 10 nm. This mean exciton migration can vary dramatically between films and within a single film due to heterogeneities in morphology on length scales of 10's to 100's nm. To study the variability of exciton diffusion and morphology within individual conjugated polymer films, we are adapting stimulated emission depletion (STED) microscopy. STED is typically used in biology with sparse well-engineered fluorescent labels or on NV-centers in diamond. I will, however, describe how we have demonstrated the extension of STED to conjugated polymer films and nanoparticles of MEH-PPV and CN-PPV, despite the presence of two photon absorption, by taking care to first understand the material's photophysical properties. We then further adapt this approach, by introducing a second ultrafast STED pulse at a variable delay. Excitons that migrate away from the initial subdiffraction excitation volume during the ps-ns time delay, are preferentially quenched by the second STED pulse, while those that remain in the initial volume survive. The resulting effect of the second STED pulse is modulated by the degree of migration over the ultrafast time delay, thus providing a new method to study exciton migration. Since this technique utilizes subdiffraction optical excitation and detection volumes with ultrafast time resolution, it provides a means of spatially and temporally resolving measurements of exciton migration on the native length and time scales. In this way, we will obtain a spatiotemporal map of exciton distributions and migration that will help to correlate the energetic landscape to film morphology at the nanoscale.

The dependence of thin-film resistivity on the thickness is known to be strongly influenced by the interaction of the conduction electrons with the surface. Great efforts have been made in recent years, mainly concerning the quantum-mechanical description of the surface scattering. Detailed discussions of this problem, however, suffer from the lack of information concerning the real topography of thin-film surfaces. The development of scanning tunneling microscopy (STM) now gives the chance of direct, quantitative imaging. In this paper, we use the topographic information of STM to improve the fitting of theoretical descriptions to the measured thickness-dependence of the resistivity. The transport parameters obtained from these calculations show a high degree of physical consistency.

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 µm spatial resolution over a large imaging area of ~24 mm(2). Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 µm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings. PMID:21935102

Atomic force microscopy of the (111) surface of macroscopic AgBr crystals revealed steps ranging in height from two atomic layers up to 10 nm, lying predominantly along the (110) and (112) families of crystal directions. Rods of elemental Ag, formed via photoreduction, were observed along the (110) family of directions. Images of adsorbed gelatin films revealed circular pores with diameters of order 10-100 nm, extending to the AgBr surface. The length of deposition time, the pH and concentration of the gelatin solution, and the presence of steps on the AgBr surface were observed to affect the size, number, and location of pores in the gelatin films. 12 refs., 7 figs.

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 μm spatial resolution over a large imaging area of ~24 mm2. Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 μm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings.

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 µm spatial resolution over a large imaging area of ~24 mm2. Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 µm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings. PMID:21935102

Anodic porous alumina (APA) films have a honeycomb cell structure of pores and a voltage-induced bi-stable switching effect. We have applied conducting atomic force microscopy (CAFM) as a method to form and to disrupt current paths in the APA films. A bi-polar switching operation was confirmed. We have firstly observed terminals of current paths as spots or areas typically on the center of the triangle formed by three pores. In addition, though a part of the current path showed repetitive switching, most of them were not observed again at the same position after one cycle of switching operations in the present experiments. This suggests that a part of alumina structure and/or composition along the current paths is modified during the switching operations.

Waveguide evanescent field fluorescence microscopy (WEFF) is a novel microscopy technology that allows imaging of a cell's plasma membrane in the vicinity of a glass substrate with high axial resolution, low background and little photobleaching. Time-lapse imaging can be performed to investigate changes in cell morphology in the presence or absence of chemical agents. WEFF microscopy provides a method to investigate plasma membranes of living cells and allows a comparison to simplified model membranes immobilized on planar substrates. The interaction of the nonionic detergent Triton X-100 with plasma membranes of osteoblasts in an aqueous environment was investigated. Solubilization of the membranes very close to the waveguide surface was visualized and related to the three-stage solubilisation model proposed for liposomes and supported lipid bilayers. Findings for the plasma membranes of cells are in excellent agreement with results reported for these artificial model systems.

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes. PMID:26333599

Electron scattering in nm-thick metal films has fundamental and technological importance. Ballistic Electron Emission Microscopy (BEEM) an STM based technique can be utilized to measure the scattering rate and understand the scattering mechanisms. By injecting electrons from the STM tip in the energy range of 0.2 eV- 1.5 eV into the metal base of a metal semiconductor diode and measuring the amount of current collected in the semiconductor a Schottky barrier height can be measured. In addition, by measuring the decay in the collector or BEEM current vs. metal film thickness, an electron attenuation length can be measured. One question has always been; what are these BEEM attenuation lengths sensitive to? Intrinsic properties of the metal, or extrinsic effects such as the structure of the film? By measuring the attenuation length of W and Cr and comparing to prior measurements of Cu, Ag, Au a comparison between the BEEM attenuation length and resistivity can be achieved over an order of magnitude in resistivity. The results show an inverse relationship that one expects for mean free path and resistivity, indicating that BEEM measurements are sensitive to the intrinsic properties of the metal and not solely the structure of the films.

In anticipation of devices scaling down further to the few nanometer regime, the ability to characterize material localized within the few nm of a critical device region poses a current challenge, particularly when the material is already buried under other material layers such as under a metal contact. Conventional techniques typically provide indirect information of the nanoscale material quality through a surface or volume averaging perspective. Here we present a study of local (nm range) oxidation in few nanometer thick Co-films using Ballistic Electron Emission Microscopy/Spectroscopy (BEEM/BEES). Co films were grown on n-Si(111) substrates, oxidized in ambient atmosphere before capping with a thin Au film to prevent further oxidation and enable BEEM measurements. In addition to BEES, the temporal progression of Co oxidation was also tracked by X-ray Photoelectron Spectroscopy. At room temperature, we report that the electron injection thresholds are sufficiently different for local regions with Co and oxidized-Co enabling their distinction in BEEM measurements. Our results demonstrate the possibility of using BEEM for nanoscale spatial mapping of the oxidized regions in Co-films, and this can provide critical information toward the successful fabrication of next generation Co-based nano-devices.

In the last years there has been a renewed interest for zinc oxide semiconductor, mainly triggered by its prospects in optoelectronic applications. In particular, zinc oxide thin films are being widely used for photovoltaic applications, in which the determination of the electrical conductivity is of great importance. Being an intrinsically doped material, the quantification of its doping concentration has always been challenging. Here we show how to probe the charge carrier density of zinc oxide thin films by Scanning Kelvin Probe Microscopy, a technique that allows measuring the contact potential difference between the tip and the sample surface with high spatial resolution. A simple electronic energy model is used for correlating the contact potential difference with the doping concentration in the material. Limitations of this technique are discussed in details and some experimental solutions are proposed. Two-dimensional doping concentration images acquired on radio frequency-sputtered intrinsic zinc oxide thin films with different thickness and deposited under different conditions are reported. We show that results inferred with this technique are in accordance with carrier concentration expected for zinc oxide thin films deposited under different conditions and obtained from resistivity and mobility measurements. PMID:24569599

Using frictional force microscopy (FFM), the friction surface characteristics were compared between twisted nematic (TN) mode and vertical alignment (VA) mode alignment films (AFs). The friction asymmetry was detected depending on temperature conditions on TN mode AF, but not on VA mode AF. The difference between two modes was explained by leaning intermolecular repulsion caused by the pre-tilt angle uniformity and the density of side chain. No level difference according to temperature conditions appeared when the pre-tilt angle were measured after liquid crystal (LC) injection.

Thin films of tetrathiafulvanene-tetracyanoquinodimethane (TTF-TCNQ) grown on mica substrates by vacuum deposition were studied by scanning tunneling microscopy (STM). STM images displayed the usual arrangement of alternative TTF and TCNQ columns aligned parallel to the crystal b axis. However, in addition to the same phase as that of a TTF-TCNQ bulk crystal, a new phase is observed. In this new phase the tilt angles the TCNQ and TTF molecular planes make with the a×b axis are different from those observed in the normal phase. This new phase can be explained by the introduction of a stacking fault on the surface.

We report a nanoscale magnetic conducting filament in a resistive random access memory (RRAM) device by the direct investigation of conducting nanobits in NiO thin films using magnetic force microscopy. The conducting nanobit in a NiO RRAM capacitor formed by CAFM and KFM exhibited a typical bistable resistive switching characteristic. The magnetizations of the conducting nanobit were measured as a function of the set-reset switching cycle and as the switching cycles were increased, a strong ferromagnetic signal was observed. The metallic Ni formation in the nanoscale magnetic conducting filament could be a possible reason for the origin of the magnetism. [Figure not available: see fulltext.

This work demonstrates implementation of low energy electron energy loss technique (EELS) in scanning transmission electron microscopy (STEM) to investigate the changes of free electron density at room temperature in ultra-thin NbN films under composite ion beam irradiation up to the deses of ∼3 d.p.a. for nitrogen atoms. It was found the constant value of the free electron density ∼1.6 ·1029 m-3 in this dose range while the irradiated material was characterized by metal type of electrical conductivity.

In this paper, a novel method that can perform measurements of the contact potential difference (CPD) between a tip and a thin film transistor (TFT) panel using the Kelvin probe force microscopy (KPFM) is proposed for inspection of critical invisible defects on TFT panels. In this application, the surface potential of a TFT panel is inferred from the electrostatic interaction force between a tip and a TFT panel induced by the electric field. The experimental results are given to illustrate that the KPFM provides a novel and feasible way to detect the most critical invisible defects on TFT panels.

We apply wide-field interferometric microscopy techniques to acquire quantitative phase profiles of ventricular cardiomyocytes in vitro during their rapid contraction with high temporal and spatial resolution. The whole-cell phase profiles are analyzed to yield valuable quantitative parameters characterizing the cell dynamics, without the need to decouple thickness from refractive index differences. Our experimental results verify that these new parameters can be used with wide field interferometric microscopy to discriminate the modulation of cardiomyocyte contraction dynamics due to temperature variation. To demonstrate the necessity of the proposed numerical analysis for cardiomyocytes, we present confocal dual-fluorescence-channel microscopy results which show that the rapid motion of the cell organelles during contraction preclude assuming a homogenous refractive index over the entire cell contents, or using multiple-exposure or scanning microscopy. PMID:21258502

In this present study we applied Raman and fluorescence microscopy to investigate the internalisation, cellular distribution and effects on cell metabolism of photosensitizer nanoparticles for photodynamic therapy in fibroblasts and macrophages.

The effect of the lubrication regime on gear performance has been recognized, qualitatively, for decades. Often the lubrication regime is characterized by the specific film thickness being the ratio of lubricant film thickness to the composite surface roughness. Three studies done at NASA to investigate gearing pitting life are revisited in this work. All tests were done at a common load. In one study, ground gears were tested using a variety of lubricants that included a range of viscosities, and therefore the gears operated with differing film thicknesses. In a second and third study, the performance of gears with ground teeth and superfinished teeth were assessed. Thicker oil films provided longer lives as did improved surface finish. These datasets were combined into a common dataset using the concept of specific film thickness. This unique dataset of more 258 tests provides gear designers with some qualitative information to make gear design decisions.

Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

Digital image analysis is a fundamental component of quantitative microscopy. However, intravital microscopy presents many challenges for digital image analysis. In general, microscopy volumes are inherently anisotropic, suffer from decreasing contrast with tissue depth, lack object edge detail, and characteristically have low signal levels. Intravital microscopy introduces the additional problem of motion artifacts, resulting from respiratory motion and heartbeat from specimens imaged in vivo. This paper describes an image registration technique for use with sequences of intravital microscopy images collected in time-series or in 3D volumes. Our registration method involves both rigid and non-rigid components. The rigid registration component corrects global image translations, while the non-rigid component manipulates a uniform grid of control points defined by B-splines. Each control point is optimized by minimizing a cost function consisting of two parts: a term to define image similarity, and a term to ensure deformation grid smoothness. Experimental results indicate that this approach is promising based on the analysis of several image volumes collected from the kidney, lung, and salivary gland of living rodents. PMID:22092443

Digital image analysis is a fundamental component of quantitative microscopy. However, intravital microscopy presents many challenges for digital image analysis. In general, microscopy volumes are inherently anisotropic, suffer from decreasing contrast with tissue depth, lack object edge detail and characteristically have low signal levels. Intravital microscopy introduces the additional problem of motion artefacts, resulting from respiratory motion and heartbeat from specimens imaged in vivo. This paper describes an image registration technique for use with sequences of intravital microscopy images collected in time-series or in 3D volumes. Our registration method involves both rigid and nonrigid components. The rigid registration component corrects global image translations, whereas the nonrigid component manipulates a uniform grid of control points defined by B-splines. Each control point is optimized by minimizing a cost function consisting of two parts: a term to define image similarity, and a term to ensure deformation grid smoothness. Experimental results indicate that this approach is promising based on the analysis of several image volumes collected from the kidney, lung and salivary gland of living rodents. PMID:22092443

The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

Control over the properties of ultrathin films plays a crucial role in many fields of science and technology. Although nondestructive optical and electrical methods have multiple advantages for local surface characterization, their applicability is very limited if the surface is in contact with an electrolyte solution. Local electrochemical methods, e.g., scanning electrochemical microscopy (SECM), cannot be used as a robust alternative yet because their methodological aspects are not sufficiently developed with respect to these systems. The recently proposed scanning electrochemical impedance microscopy (SEIM) can efficiently elucidate many key properties of the solid/liquid interface such as charge transfer resistance or interfacial capacitance. However, many fundamental aspects related to SEIM application still remain unclear. In this work, a methodology for the interpretation of SEIM data of "charge blocking systems" has been elaborated with the help of finite element simulations in combination with experimental results. As a proof of concept, the local film thickness has been visualized using model systems at various tip-to-sample separations. Namely, anodized aluminum oxide (Al2O3, 2-20 nm) and self-assembled monolayers based on 11-mercapto-1-undecanol and 16-mercapto-1-hexadecanethiol (2.1 and 2.9 nm, respectively) were used as model systems. PMID:26871004

Finds that exposure to graphic photographs of worms taken from a horror film increased children's enjoyment of the horror movie segment and reduced fear reactions to the scene. Shows that exposure to a live earthworm was effective in reducing fear reactions to the movie only among boys but did alter children's affective reactions to and judgments…

The existence of notochord distinguishes chordates from other phyla. Amphioxus is the only animal that keeps notochord during the whole life. Notochord is a unique organ for amphioxus, with its vertically arranged muscular notochordal plates, which is different from notochords in embryos of other chordates. We use stimulated Raman scattering (SRS) microscopy as a non-invasive technique to image the chemical components in amphioxus notochord. SRS provides chemical specificity as spontaneous Raman does and offers a higher sensitivity for fast acquisition. Unlike coherent anti- Stokes Raman scattering (CARS) microscopy, SRS microscopy doesn't have non-resonant background and can better differentiate different components in the specimen. We verify that the notochord is a protein-rich organ, which agrees well with the result of conventional staining methods. Detailed structures in notochordal plates and notochordal sheath are revealed by SRS microscopy with diffraction limited resolution. Our experiment shows that SRS microscopy is an excellent imaging tool for biochemical research with its intrinsic chemical selectivity, high spatiotemporal resolution and native 3D optical sectioning ability.

The influence of oxygen partial pressure on the Fermi level of ZnO films prepared by pulsed laser deposition is investigated. The contact potential difference of the ZnO films fabricated under various oxygen partial pressures is studied systematically using Kelvin probe force microscopy. The Fermi level shifted by 0.35 eV as oxygen partial pressure increased. This indicates a significant change in the electronic structure and energy balance in ZnO films. This fact provides a consistent explanation that the changes in carrier concentration, resistivity and mobility of ZnO films are attributed to oxygen vacancy induced shift of the Fermi level.

We obtained the NMR spectrum and the spin-lattice relaxation time (T1) for thin film samples by magnetic resonance force microscopy (MRFM). The samples were CaF2 thin films which were 50 nm and 150 nm thick. T1 was measured at 18 K using a cyclic adiabatic inversion method at a fixed frequency. A comparison of the bulk and two thin films showed that T1 becomes shorter as the film thickness decreases. To make the comparison as accurate as possible, all three samples were loaded onto different beams of a multi-cantilever array and measured in the same experimental environment. PMID:25828244

We obtained the NMR spectrum and the spin-lattice relaxation time (T1) for thin film samples by magnetic resonance force microscopy (MRFM). The samples were CaF2 thin films which were 50 nm and 150 nm thick. T1 was measured at 18 K using a cyclic adiabatic inversion method at a fixed frequency. A comparison of the bulk and two thin films showed that T1 becomes shorter as the film thickness decreases. To make the comparison as accurate as possible, all three samples were loaded onto different beams of a multi-cantilever array and measured in the same experimental environment.

Collagen is the major fibrous protein in the extracellular matrix and consists a significant component of skin, bone, cartilage and tendon. Due to its unique properties, it has been widely used as scaffold or culture substrate for tissue regeneration or/and cell-substrate interaction studies. The ultraviolet light-collagen interaction investigations are crucial for the improvement of many applications such as that of the UV irradiation in the field of biomaterials, as sterilizing and photo-cross-linking method. The aim of this paper was to investigate the mechanisms of UV-collagen interactions by developing a collagen-based, well characterized, surface with controlled topography of collagen thin films in the nanoscale range. The methodology was to quantify the collagen surface modification induced on ultraviolet radiation and correlate it with changes induced in cells. Surface nanoscale characterization was performed by Atomic Force Microscopy (AFM) which is a powerful tool and offers quantitative and qualitative information with a non-destructive manner. In order to investigate cells behavior, the irradiated films were used for in vitro cultivation of human skin fibroblasts and the cells morphology, migration and alignment were assessed with fluorescence microscopy imaging and image processing methods. The clarification of the effects of UV light on collagen thin films and the way of cells behavior to the different modifications that UV induced to the collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist the appropriate use of UV light for developing biomaterials.

The tribological and rheological behavior of thin polymer films at the nanometer length scale has become a topic of extreme technological and scientific interest. The friction and wear characteristics of ultrathin organic coatings are critical in magnetic storage media devices, as well as emerging technologies such as microelectromechanical devices. In the microelectronics industry, the ability to produce ultrathin coatings of photoresists and electron resists that are free of scratches and thickness fluctuations is a crucial step in any lithography process. Fortunately the need to understand the behavior of ultrathin organic films has coincided with the development of the scanning probe microscope (SPM) which is able to impose shear and tensile forces, and image the resulting deformations, on the nanometer scale. In contrast to traditional scientific disciplines like condensed matter physics and physical chemistry, the "nanoscience" community has only recently begun to examine the role of temperature in material response. This is largely because piezoelectric transducers are incompatible with substantial temperature elevation. A recent advance in SPM design has isolated the transducer and accompanying electronics from the sample, enabling investigators to heat samples to temperatures as high as 170°C without affecting the performance of the instrument. Using an environmental SPM, we examined the temperature and rate dependence of tip-imposed plastic and viscoelastic deformations in thin polymer films. Viscous flow in defects in nonwetting films was investigated as well. Chapter 1 provides a brief review of viscoelastic and plastic deformations in bulk polymers, the glass transition temperature, and the effect of confining polymer molecules to an interface on the observed glass transition temperature. Chapter 2 discusses scanning probe microscopy instrumentation, techniques, and applications to polymer thin film tribology. In Chapters 3 and 4, results are

RNA interference (RNAi) is a powerful technique enabling the identification of the genes involved in a certain cellular process. Here, we discuss protocols for microscopy-based RNAi screening in protonemal cells of the moss Physcomitrella patens, an emerging model system for plant cell biology. Our method is characterized by the use of conditional (inducible) RNAi vectors, transgenic moss lines in which the RNAi vector is integrated, and time-lapse fluorescent microscopy. This method allows for effective and efficient screening of >100 genes involved in various cellular processes such as mitotic cell division, organelle distribution, or cell growth. PMID:27581297

We apply electrostatic force microscopy (EFM) to study defects in GaAs films grown on Ge. On a GaAs film with surface antiphase boundaries (APBs), we reproducibly measure the surface contact potential (SCP) at the APBs to be (30{plus_minus}5) mV higher than that of the domains, due to the surface Fermi level at APBs being pinned closer to the valence band maximum. On a thick film which contains buried APBs and wedge-shaped depressions on the surface, we find that the SCP of the wedge-shaped depressions is (25{plus_minus}5) mV lower than that of the GaAs surface. Hence, these wedge-shaped depressions have defect electronic states different from those of APBs. The capacitance gradient ({partial_derivative}C/{partial_derivative}z) contrasts on the two samples are also shown to arise from different origins. Factors that can affect the measured SCP and {partial_derivative}C/{partial_derivative}z values are discussed. We demonstrate a new application of EFM to distinguish different types of defects by measuring variations in relative SCP (thus the work function or position of Fermi level) and/or {partial_derivative}C/{partial_derivative}z on sample surfaces. The spatial resolutions of SCP and {partial_derivative}C/{partial_derivative}z are 30 nm, limited by the tip size. {copyright} {ital 1999 American Institute of Physics.}

High-resolution transmission electron microscopy (HRTEM) has been used to investigate the microstructure of diamond films grown by plasma-assisted chemical vapor deposition using fullerene precursors. HRTEM observations of as-grown films revealed an array of larger crystals (>200 nm) within a polycrystalline matrix of much smaller crystallites (<20 nm). The randomly oriented small crystallites were nearly free of structural imperfections such as stacking faults or twins, while the larger ones had preferred <110> orientations with respect to the Si (100) substrate and showed evidence of structural defects on the periphery of the crystals. The most common defects were V-shaped {Sigma}9 twin boundaries, which are generally believed to serve as re-entrant sites for diamond nucleation and growth. The observation of growth steps on both (111) and (110) surfaces seems to support a reaction model in which fragments of C{sub 60}, including C{sub 2}, are considered the growth species. In particular, the nanocrystallinity of the films is most likely due to a high carbon cluster density from C{sub 60} fragmentation at or near the diamond surface, which can serve as nucleation sites for the growth of new crystallites.

The dynamic analysis of biological living samples is one of the particular interests in life sciences. An improved digital holographic microscope for long-term quantitative phase-contrast imaging of living cells is presented in this paper. The optical configuration is optimized in the form of a free-space-fiber hybrid system which promotes the flexibility of imaging in complex or semi-enclosed experimental environment. Aberrations compensation is implemented taking into account the additional phase aberration induced by liquid culture medium in long-term observation. The proposed approach is applied to investigate living samples of MC3T3-E1 and MLO-Y4 cells. The experimental results demonstrate its availability in the analysis of cellular changes.

Detection for clinical diagnosis and study of microbial cell is performed by a combination of low magnification optical microscopy and direct and indirect labeling techniques. Visual ultrastructural studies on subcellular organelles are possible with variations of electron microscopy (thin section, scanning and freeze fracture), although specimen preparation steps such as fixation, dehydration, resin embedding, ultra-thin sectioning, coating and staining are very specialized, extensive and may introduce artifacts in the original sample. The development of high resolution x-ray microscopy is a new technique well suited to observe the intact structure of a biological specimen at high resolution without any artifacts. Here, x ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichia coli, and Lipopolysaccharide from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50 nm spatial resolutions are achieved and images are able to be discussed on the biological view.

Soft x-ray contact microscopy provides the biologist with a technique for examining the ultrastructure of living cells at a much higher resolution than that possible by various forms of light microscopy. Readout of the developed photoresist using atomic force microscopy (AFM) produces a detailed map of the carbon densities generated in the resist following exposure of the specimen to water-window soft x-rays (2--4nm) produced by impact of a high energy laser onto a suitable target. The established high resolution imaging method of transmission electron microscopy (TEM) has inherent problems in the chemical pre-treatment required for producing the ultrathin sections necessary for this technique. Using the unicellular green alga Chlamydomonas the ultrastructural appearance of the cells following SXCM and TEM has been compared. While SXCM confirms the basic structural organization of the cell as seen by TEM (e.g., the organization of the thylakoid membranes within the chloroplast; flagellar insertion into the cytoplasm), there are important differences. These are in the appearance of the cell covering and the presence of carbon-dense spherical cellular inclusions.

Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes. PMID:26643278

I present multimodal wide-field interferometric microscopy platform for label-free 3-D imaging of live cells during fast flow. Using holographic optical tweezers, multiple cells can be optically trapped and rapidity rotated on all axes, while acquired using an external off-axis wide-field interferometric module developed in our lab. The interferometric projections are rapidly processed into the 3-D refractive-index profile of the cells using a tomographic phase microscopy algorithms that take into consideration optical diffraction effects. The algorithms for the 3-D refractive-index reconstruction, and for calculating various morphological parameters that should serve for online sorting of cells, are efficiently implemented in a nearly real-time manner. The potential of this new high-throughput imaging technique is for label-free image analysis and sorting of cells during flow, to substitute current cell sorting devices, which are based on external labeling that eventually damages the cell sample.

We present an efficient and fast method for selective and localized electroporation of a single living cell from a population of millions to tens of cells using the modified tip of an atomic force microscope. Electroporation was observed in real time using an inverted microscope. This technique is proposed as a tool for efficient and controlled delivery of biomolecules, proteins, drugs, and genes.

Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts. PMID:25861930

Scanning electrochemical microscopy (SECM) is increasingly applied to study and image live cells. Quantitative analyses of biological systems, however, still remain challenging. In the presented study, single human adenocarcinoma cervical cancer cells are electrochemically investigated by means of SECM. The target cell's electrochemical response is observed over time under the influence of green tea catechins (GTC), which are suggested to offer chemopreventive and therapeutic effects on cancer. The electrochemical response of living target cells is measured experimentally and quantified in an apparent heterogeneous rate constant by using a numerical model, based on forced convection during high speed SECM imaging. The beneficial effect of GTC on cancer cells could be confirmed by SECM, and the presented study shows an alternative approach toward unraveling the mechanisms involved during inhibition of carcinogenesis. PMID:26167832

We demonstrate the potential to modify the magnetic behavior and structural properties of ferromagnetic thin films using focused ion beam 'direct-write' lithography. Patterns inspired by the split-ring resonators often used as components in meta-materials were defined upon 15 nm Fe films using a 30 keV Ga{sup +} focused ion beam at a dose of 2 x 10{sup 16} ions cm{sup -2}. Structural, chemical and magnetic changes to the Fe were studied using transmission soft X-ray microscopy at the ALS, Berkeley CA. X-ray absorption spectra showed a 23% reduction in the thickness of the film in the Ga irradiated areas, but no chemical change to the Fe was evident. X-ray images of the magnetic reversal process show domain wall pinning around the implanted areas, resulting in an overall increase in the coercivity of the film. Transmission electron microscopy showed significant grain growth in the implanted regions.

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

Copper is widely used material for electrical interconnects within integrated circuits and recently as a base layer for hot electron spin injection and readout into silicon. Integral to both their applications is the knowledge of the electron scattering length. To the best of our knowledge, little work exists that directly measures the scattering length of electrons in copper. In this study we used ballistic electron emission microscopy (BEEM) to measure the hot electron attenuation length of copper thin films deposited on Si(001). BEEM is a three terminal scanning tunneling microcopy (STM) based technique that can measure transport and Schottky heights of metal/semiconductor systems. We find a Schottky height of 0.67 eV and an attenuation length approaching 40 nm just above the Schottky height at 77 K. We also measure a decrease in the attenuation length with increasing tip bias to determine the relative roles of inelastic and elastic scattering.

Charge carrier trapping degrades the performance of organometallic halide perovskite solar cells. To characterize the locations of electronic trap states in a heterogeneous photoactive layer, a spatially resolved approach is essential. Here, we report a comparative study on methylammonium lead tri-iodide perovskite thin films subject to different thermal annealing times using a combined photoluminescence (PL) and femtosecond transient absorption microscopy (TAM) approach to spatially map trap states. This approach coregisters the initially populated electronic excited states with the regions that recombine radiatively. Although the TAM images are relatively homogeneous for both samples, the corresponding PL images are highly structured. Themore » remarkable variation in the PL intensities as compared to transient absorption signal amplitude suggests spatially dependent PL quantum efficiency, indicative of trapping events. Furthermore, detailed analysis enables identification of two trapping regimes: a densely packed trapping region and a sparse trapping area that appear as unique spatial features in scaled PL maps.« less

Charge carrier trapping degrades the performance of organometallic halide perovskite solar cells. To characterize the locations of electronic trap states in a heterogeneous photoactive layer, a spatially resolved approach is essential. Here, we report a comparative study on methylammonium lead tri-iodide perovskite thin films subject to different thermal annealing times using a combined photoluminescence (PL) and femtosecond transient absorption microscopy (TAM) approach to spatially map trap states. This approach coregisters the initially populated electronic excited states with the regions that recombine radiatively. Although the TAM images are relatively homogeneous for both samples, the corresponding PL images are highly structured. The remarkable variation in the PL intensities as compared to transient absorption signal amplitude suggests spatially dependent PL quantum efficiency, indicative of trapping events. Detailed analysis enables identification of two trapping regimes: a densely packed trapping region and a sparse trapping area that appear as unique spatial features in scaled PL maps. PMID:27103096

Copper and gold films (0.2 to 2 microns) were ion plated onto polished 304-stainless-steel surfaces. These coatings were examined by scanning electron microscopy for coating growth defects. Three types of defects were distinguished: nodular growth, abnormal or runaway growth, and spits. The cause and origin for each type of defect was traced. Nodular growth is primarily due to inherent substrate microdefects, abnormal or runaway growth is due to external surface inclusions, and spits are due to nonuniform evaporation. All these defects have adverse effects on the coatings. They induce stresses and produce porosity in the coatings and thus weaken their mechanical properties. Friction and wear characteristics are affected by coating defects, since the large nodules are pulled out and additional wear debris is generated.

Microspectrofluorometric measurements are made to determine uptake and distribution of oil in marine organisms after exposure to crude oil. Equipment combining fluorescence microscopy with spectral analysis of the fluorescence emission is described. After contamination with oil, the intestine content of Lumbricillus lineatus, Nereis diversicolor and Anaitides mucosa shows a fluorescence emission at long wavelengths with a maximum at about 550 nm; this is in contrast to the fluorescence emission of these organisms without oil contamination. There is evidence that aromatic hydrocarbons are metabolized in the intestine of the worms studied.

While biochemical pathways within mitotic cells have been intensively studied, the mechanics of dividing cells is only poorly understood. In our recent report, an experimental system combining fluorescence and atomic force microscopy was set up to study dynamics of mitotic rounding of mammalian cells. We show that cells have a rounding pressure that increases upon mitotic entry. Using specific inhibitors or perturbations, we revealed biological processes required for force generation that underpin the cell rounding shape change during mitosis. The significance of the finding and an outlook are discussed.

The emergence of efficient hybrid organic-inorganic perovskite photovoltaic materials has caused the rapid development of a variety of preparation and processing techniques designed to maximize their performance. As processing methods continue to emerge, it is important to understand how the optical properties of these materials are affected on a microscopic scale. Here polarization resolved two-photon total internal reflectance microscopy (TIRFM) was used to probe changes in transition dipole moment orientation as a function of thermal annealing time in hybrid organic-inorganic lead iodide based perovskite (CH3NH3PbI3) thin films on glass. These results show that as thermal annealing time is increased the distribution of transition moments pointing out-of-plane decreases in favor of forming areas with increased in-plane orientations. As a result, it was also shown through the axial sensitivity of TIRFM that the surface topography is manifested in the signal intensity and can be used to survey aspects of morphology in coincidence with the optical properties of these films.

Using ultrahigh-vacuum low-temperature scanning tunneling microscopy and spectroscopy combined with first principles density functional theory calculations, we have investigated structural and electronic properties of pristine and potassium (K)-deposited picene thin films formed in situ on a Ag(111) substrate. At low coverages, the molecules are uniformly distributed with the long axis aligned along the [112̄] direction of the substrate. At higher coverages, ordered structures composed of monolayer molecules are observed, one of which is a monolayer with tilted and flat-lying molecules resembling a (11̄0) plane of the bulk crystalline picene. Between the molecules and the substrate, the van der Waals interaction is dominant with negligible hybridization between their electronic states; a conclusion that contrasts with the chemisorption exhibited by pentacene molecules on the same substrate. We also observed a monolayer picene thin film in which all molecules were standing to form an intermolecular π stacking. Two-dimensional delocalized electronic states are found on the K-deposited π stacking structure. PMID:25240362

Using ultrahigh-vacuum low-temperature scanning tunneling microscopy and spectroscopy combined with first principles density functional theory calculations, we have investigated structural and electronic properties of pristine and potassium (K)-deposited picene thin films formed in situ on a Ag(111) substrate. At low coverages, the molecules are uniformly distributed with the long axis aligned along the [112{sup ¯}] direction of the substrate. At higher coverages, ordered structures composed of monolayer molecules are observed, one of which is a monolayer with tilted and flat-lying molecules resembling a (11{sup ¯}0) plane of the bulk crystalline picene. Between the molecules and the substrate, the van der Waals interaction is dominant with negligible hybridization between their electronic states; a conclusion that contrasts with the chemisorption exhibited by pentacene molecules on the same substrate. We also observed a monolayer picene thin film in which all molecules were standing to form an intermolecular π stacking. Two-dimensional delocalized electronic states are found on the K-deposited π stacking structure.

We study planar defects in epitaxial Co:ZnO dilute magnetic semiconductor thin films deposited on c-plane sapphire (Al{sub 2}O{sub 3}), as well as the Co:ZnO/Al{sub 2}O{sub 3} interface, using aberration-corrected transmission electron microscopy and electron energy-loss spectroscopy. Co:ZnO samples that were deposited using pulsed laser deposition and reactive magnetron sputtering are both found to contain extrinsic stacking faults, incoherent interface structures, and compositional variations within the first 3–4 Co:ZnO layers next to the Al{sub 2}O{sub 3} substrate. The stacking fault density is in the range of 10{sup 17} cm{sup −3}. We also measure the local lattice distortions around the stacking faults. It is shown that despite the relatively high density of planar defects, lattice distortions, and small compositional variation, the Co:ZnO films retain paramagnetic properties.

A number of practical issues must be addressed when using thin carbon films as quarter-wave plates for Zernike phase-contrast electron microscopy. We describe, for example, how we meet the more stringent requirements that must be satisfied for beam alignment in this imaging mode. In addition we address the concern that one might have regarding the loss of some of the scattered electrons as they pass through such a phase plate. We show that two easily measured parameters, (1) the low-resolution image contrast produced in cryo-EM images of tobacco mosaic virus particles and (2) the fall-off of the envelope function at high resolution, can be used to quantitatively compare the data quality for Zernike phase-contrast images and for defocused bright-field images. We describe how we prepare carbon-film phase plates that are initially free of charging or other effects that degrade image quality. We emphasize, however, that even though the buildup of hydrocarbon contamination can be avoided by heating the phase plates during use, their performance nevertheless deteriorates over the time scale of days to weeks, thus requiring their frequent replacement in order to maintain optimal performance. PMID:19157711

The emergence of efficient hybrid organic-inorganic perovskite photovoltaic materials has caused the rapid development of a variety of preparation and processing techniques designed to maximize their performance. As processing methods continue to emerge, it is important to understand how the optical properties of these materials are affected on a microscopic scale. Here polarization resolved two-photon total internal reflectance microscopy (TIRFM) was used to probe changes in transition dipole moment orientation as a function of thermal annealing time in hybrid organic-inorganic lead iodide based perovskite (CH3NH3PbI3) thin films on glass. These results show that as thermal annealing time is increased themore » distribution of transition moments pointing out-of-plane decreases in favor of forming areas with increased in-plane orientations. As a result, it was also shown through the axial sensitivity of TIRFM that the surface topography is manifested in the signal intensity and can be used to survey aspects of morphology in coincidence with the optical properties of these films.« less

Structural characterization by super-resolution microscopy has become increasingly widespread, particularly in the biological community. The technique is powerful because it can produce real-space images with resolutions of tens of nanometers, while sample preparation is relatively non-invasive. Previous studies have applied these techniques to important scientific problems in the life sciences, but relatively little work has explored the attainable limit of resolution using samples of known structure. In this work, we apply photo-activated localization microscopy (PALM) to polymer films that have been nanopatterned using electron-beam lithography. Trace amounts of a rhodamine spiroamide dye are dispersed into nanostructured poly(methyl methacrylate), and UV-induced switching of the fluorophores enables nanoscale localization of single molecules to generate a final composite super-resolution image. Features as small as 50 nm are clearly resolvable. To determine the ultimate resolution limit, we investigate sources of error in the system, particularly from systematic mislocalizations due to the effect of fluorophore orientation on the single-molecule point-spread function.

A high-resolution in situ spectroelectrochemical optical diffraction experiment has been developed to understand the volume expansion/contraction process of amorphous silicon (a-Si) thin-film anodes. Electrodes consisting of 1D transmissive gratings of silicon have been produced through photolithographic methods. After glovebox assembly in a home-built Teflon cell, monitoring of the diffraction efficiency of these gratings during the lithiation/delithiation process is performed using an optical microscope equipped with a Bertrand lens. When the diffraction efficiency along with optical constants obtained from in situ spectroscopic ellipsometry is utilized, volume changes of the active materials can be deduced. Unlike transmission electron microscopy and atomic force microscopy characterization methods of observing silicon's volume expansion, this experiment allows for real-time monitoring of the volume change at charge/discharge cycles greater than just the first few along with an experimental environment that directly mimics that of a real battery. This technique shows promising results that provide needed insight into understanding the lithium alloying reaction and subsequent induced capacity fade during the cycling of alloying anodes in lithium-ion batteries. PMID:27311132

Amorphous/nanocrystalline SiOx/CdSe, GeS2/CdSe, SiOx/ZnSe and Se/CdSe amorphous multilayers (MLs) were grown by consecutive physical vapour deposition of the constituent materials at room substrate temperature. A step-by-step manner of deposition was applied for the preparation of each layer (2 10nm thick) of MLs. Surface morphology has been investigated by atomic force microscopy (AFM) in order to get information about ML interfaces. For a scanned area of 3.4×4μmSiOx/CdSe and GeS2/CdSe MLs showed surface roughness which is around three times greater than the roughness of SiOx/ZnSe MLs. This observation has been connected with effects of both film composition and deposition rate. For a scanned area of 250×250nm the roughness determined in all MLs displayed close values and a similar increase with the ML period. The latter has been related to the flexible structure of amorphous materials. The AFM results, in good agreement with previous X-ray diffraction and high resolution electron microscopy data, indicate that the application of step-by-step physical vapour deposition makes possible fabrication of various amorphous/nanocrystalline MLs with smooth interfaces and good artificial periodicity at low substrate temperatures.

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis. PMID:24525751

Multifunctional oxide thin-films grown on silicon and several oxide substrates have been characterized using High Resolution (Scanning) Transmission Electron Microscopy (HRTEM), Energy-Dispersive X-ray Spectroscopy (EDX), and Electron Energy-Loss Spectroscopy (EELS). Oxide thin films grown on SrTiO3/Si pseudo-substrate showed the presence of amorphised SrTiO3 (STO) at the STO/Si interface. Oxide/oxide interfaces were observed to be atomically clean with very few defects. Al-doped SrTiO3 thin films grown on Si were of high crystalline quality. The Ti/O ratio estimated from EELS line scans revealed that substitution of Ti by Al created associated O vacancies. The strength of the crystal field in STO was measured using EELS, and decreased by ~1.0 eV as Ti4+ was substituted by Al3+. The damping of O-K EELS peaks confirmed the rise in oxygen vacancies. For Co-substituted STO films grown on Si, the EDS and EELS spectra across samples showed Co doping was quite random. The substitution of Ti4+ with Co3+ or Co2+ created associated oxygen vacancies for charge balance. Presence of oxygen vacancies was also confirmed by shift of Ti-L EELS peaks towards lower energy by ~0.4 eV. The crystal-field strength decreased by ~0.6 eV as Ti4+ was partially substituted by Co3+ or Co2+. Spinel Co3O4 thin films grown on MgAl2O 4 (110) were observed to have excellent crystalline quality. The structure of the Co3O4/MgAl2O4 interface was determined using HRTEM and image simulations. It was found that MgAl 2O4 substrate is terminated with Al and oxygen. Stacking faults and associated strain fields in spinel Co3O4 were found along [111], [001], and [113] using Geometrical Phase Analysis. NbO2 films on STO (111) were observed to be tetragonal with lattice parameter of 13.8 A and NbO films on LSAT (111) were observed to be cubic with lattice parameter of 4.26 A. HRTEM showed formation of high quality NbOx films and excellent coherent interface. HRTEM of SrAl4 on LAO (001) confirmed an island

In force modulation atomic force microscopy (FMAFM), vertical oscillation of the scanning tip of the AFM is added purposely and the deflection of the tip, which is influenced by surface features of the sample, is used as the z dimension to construct images. FMAFM represents a powerful technique for scientific research, but its merit has not been realized adequately to date. In this paper, the basic principles and particular features, as well as potential drawbacks of the technique, are presented and demonstrated systematically, through its application to electrochemically deposited cerium oxide films. Comparisons are also made with the more familiar contact mode AFM (CMAFM) and tapping mode AFM (TMAFM). It is shown that FMAFM reveals the major topographic features of CMAFM, but affords (i) greater resolution for sample features that are difficult in CMAFM, and (ii) continuous two-dimensional mapping of local mechanical properties on a scale of nanometres that the CMAFM, TMAFM and any other techniques, are not capable of sensing. This information can be used to elucidate other properties of the investigated surface, such as crystallinity variation, phase separation and distribution, and mechanisms of formation of deposited films. Major artifacts associated with the technique include `wedge cavity effect' and `tip slip effect', for which a geometric model is proposed to elucidate their origins. The cerium oxide films are shown to be composed of relatively hard crystalline grains, of well-defined individual geometry and comparatively regular packing, alongside relatively soft amorphous patches, devoid of distinct geometry and assembled disorderly. These features are consistent with a nucleation and growth mechanism of the deposition, in which crystalline nuclei arise and grow from an intermediate cerium gel mass, produced in the interfacial region during deposition.

Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2- transients and oscillations. Decreasing the power of the laser light decreased the percent-age of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was approximately 90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+ oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells. PMID:12661552

Herein, time-dependent scanning Kelvin probe microscopy of solution processed organic thin film transistors (OTFTs) reveals a correlation between film microstructure and OTFT device performance with the location of trapped charge within the device channel. The accumulation of the observed trapped charge is concurrent with the decrease in I{sub SD} during operation (V{sub G}=-40 V, V{sub SD}= -10 V). We discuss the charge trapping and dissipation dynamics as they relate to the film structure and show that application of light quickly dissipates the observed trapped charge.

Photoelectron emission microscopy (PEEM) and differential (optical) reflectance spectroscopy (DRS) have proven independently to be versatile analytical tools for monitoring the evolution of organic thin films during growth. In this paper, we present the first experiment in which both techniques have been applied simultaneously and synchronously. We illustrate how the combined PEEM and DRS results can be correlated to obtain an extended perspective on the electronic and optical properties of a molecular film dependent on the film thickness and morphology. As an example, we studied the deposition of the organic molecule α-sexithiophene on Ag(111) in the thickness range from submonolayers up to several monolayers. PMID:26523159

We present a dark field microscopy for the measurement of film surface roughness. The underlying principle is that a darker region in a film surface represents a deeper place from the top surface. The performance of the imaging system was demonstrated for silicon nitride films deposited in a SiH4-NH3 plasma. The system provided distributions of particles and the tendency of particle counts was compared with that of AFM-measured surface roughness. A strong correlation identified between them represents that the system is a viable alternative to AFM. The system is expected to find applications to roughness measurement during a real-time plasma processes. PMID:25942839

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines. PMID:25140415

An auxin/IAA induced in vivo green fluorescent protein (GFP) in a living plant Arabidopsis root has been studied by a scanning near-field microscope in transmission mode. The promising near-field images of the inducible GFPs at sub- surface of a plant cell suggest that they may locate proximity to the cell wall, i.e. both sides of and in the cytoplasm membrane. The clear and faint fluorescent spots with 1-3 micrometers showed that the proteins localized nearer and farther to the cell wall, respectively. All GFP molecules gathered together in a cell, and no individual GFP was observed in the experiment.

The mechanical properties of SiC films grown via C{sub 60} precursors were determined using atomic force microscopy (AFM). Conventional silicon nitride and modified diamond cantilever AFM tips were employed to determine the film hardness, friction coefficient, and elastic modulus. The hardness is found to be between 26 and 40 GPa by nanoindentation of the film with the diamond tip. The friction coefficient for the silicon nitride tip on the SiC film is about one third that for silicon nitride sliding on a silicon substrate. By combining nanoindentation and AFM measurements an elastic modulus of {approximately}300 GPa is estimated for these SiC films. In order to better understand the atomic scale mechanisms that determine the hardness and friction of SiC, we simulated the molecular dynamics of a diamond indenting a crystalline SiC substrate.

The travel of droplets, bubbles, vesicles, capsules, living cells or small organisms in microchannels is a hallmark in microfluidics applications. A full description of the dynamics of such objects requires a quantitative understanding of the complex hydrodynamic and interfacial interactions between objects and channel walls. In this paper, we present an interferometric method that allows absolute topographic reconstruction of the interspace between an object and channel walls for objects confined in microfluidic channels. Wide field microscopic imaging in reflection interference contrast mode (RICM) is directly performed at the bottom wall of microfluidic chips. Importantly, we show that the reflections at both the lower and upper surface of the microchannel have to be considered in the quantitative analysis of the optical signal. More precisely, the contribution of the reflection at the upper surface is weighted depending on the light coherence length and channel height. Using several wavelengths and illumination apertures, our method allows reconstructing the topography of thin films on channel walls in a range of 0-500 nm, with a precision as accurate as 2 nm for the thinnest films. A complete description of the protocol is exemplified for oil in water droplets travelling in channels of height 10-400 μm at a speed up to 5 mm s(-1). PMID:26830018

The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle's hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

SUMMARY Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state, and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, direct observation of structural details of infecting HIV-1 has not been realized due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by developing a correlative high-speed 3D live-cell imaging and cryoET method. Using this methodology, we showed, for the first time under near-native conditions, that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host-cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantage of our correlative live-cell and cryoET approach to image dynamic processes, such as viral infection. PMID:22078557

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle’s hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm. PMID:25222297

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm. PMID:25222297

Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

Application of atomic force microscopy (AFM) to biological objects and processes under physiological conditions has been hampered so far by the deformation and destruction of the soft biological materials invoked. Here we describe a new mode of operation in which the standard V-shaped silicon nitride cantilever is oscillated under liquid and damped by the interaction between AFM tip and sample surface. Because of the viscoelastic behavior of the cellular surface, cells effectively "harden" under such a tapping motion at high frequencies and become less susceptible to deformation. Images obtained in this way primarily reveal the surface structure of the cell. It is now possible to study physiological processes, such as cell growth, with a minimal level of perturbation and high spatial resolution (approximately 20 nm). PMID:7819507

The "two photon absorption" phenomenon had been predicted by the American Physicist, Maria Ghöppert-Mayer in 1931. Denk and Webb group had proved it in 1990 and the first product had been launched in the market in 1996. But ever since the product became available, the number of users are not increased. Moreover, the system had been too difficult to use and the system sometimes stay not working in labs. But recently, the new easier-to-use products are released and the ultra short pulse IR laser became stable. And its applications are extending from neuro-science to oncology or immunology fields. Due to these reasons, the shipment of multi-photon microscope in Japan in 2013 is approximately 40 units which is 3 times bigger than in 2010. In this paper, I would like to discuss the principles of two-photon microscopy and some of the new technologies for the higher signal capture efficiency. PMID:26017864

Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrated various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in bright-field and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multispot focus measurements, and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions. PMID:19658173

This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

Space exploration is the subject of intense media interest in a way unparalleled in any other branch of science. It is the subject of countless films and television programmes, both fact and fiction, many using original footage from space. Astronauts have broadcast live from the Moon, and TV journalists have travelled to Mir, similar to the use of exotic terrestrial locations for filming by professional film crews. Although prohibitively expensive at the moment, the next generation of spacecraft may lower launch costs to an affordable level, so that space locations become competitive against computer graphics and model work. The construction of orbital hotels will create the demand for human interest stories similar to those set in holiday locations like the south of France and Italy made just after the Second World War, at a time when mass tourism on foreign holidays was just beginning, aided by the development of large transport aircraft able to cater to the demand for mass flight.

Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges--the most evident of which are spatial limitations--to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and--being designed almost exclusively for light microscopy or electrophysiology studies--seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker's series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig's continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development. PMID:26666980

Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development.

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration. PMID:26732243

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration. PMID:26732243

Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development. PMID:26666980

Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

The personal and social costs of HIV are well documented. What remains unknown is the effect of public disclosure of HIV status on the individual who is doing the disclosing. This study describes the experience of four African American women living with HIV who participated in the development of an intergenerational education intervention for African American adolescent girls. These women suggested that they be filmed discussing the "dark side" of HIV in an effort to create an intergenerational education intervention that would alter the risk-taking behavior that they observed in young women in their community. After a rough cut of the film was completed, these women viewed the film and participated in a focus group during which they discussed what it was like to reveal and revisit their own painful experiences associated with becoming infected and then living with HIV. Findings from content analysis of transcribed dialogue included the following positive themes: (a) self-acceptance by telling one's own story and hearing the stories of the other women, (b) a sense of liberation by disclosing publicly one's image and message and letting go of others' judgments, (c) feeling supported by meeting other women who share the same experience, (d) value of using the film to impact or save young people from the pain one has experienced. A negative theme emerged related to personal pain in reliving the individual's history with HIV. PMID:16438124

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Calpha), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658

To improve our understanding of lipid metabolism, Drosophila is used as a model animal, and its lipid homeostasis is monitored by coherent anti-Stokes Raman scattering microscopy. We are able to achieve in vivo imaging of larval fat body (analogous to adipose tissue in mammals) and oenocytes (analogous to hepatocytes) in Drosophila larvae at subcellular level without any labeling. By overexpressing two lipid regulatory proteins-Brummer lipase (Bmm) and lipid storage droplet-2 (Lsd-2)-we found different phenotypes and responses under fed and starved conditions. Comparing with the control larva, we observed more lipid droplet accumulation by ˜twofold in oenocytes of fat-body-Bmm-overexpressing (FB-Bmm-overexpressing) mutant under fed condition, and less lipid by ˜fourfold in oenocytes of fat-body-Lsd-2-overexpressing (FB-Lsd-2-overexpressing) mutant under starved condition. Moreover, together with reduced size of lipid droplets, the lipid content in the fat body of FB-Bmm-overexpressing mutant decreases much faster than that of the control and FB-Lsd-2-overexpressing mutant during starvation. From long-term starvation assay, we found FB-Bmm-overexpressing mutant has a shorter lifespan, which can be attributed to faster consumption of lipid in its fat body. Our results demonstrate in vivo observations of direct influences of Bmm and Lsd-2 on lipid homeostasis in Drosophila larvae.

Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions. PMID:26567131

Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing loss are commonly available for research. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes the study of their hair cells difficult. We developed a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae were harvested, left intact within their otic capsule bone, and glued upright in a recording chamber. The bone overlying the region of the cochlear epithelium to be studied was opened and Reissner's membrane was incised. A fluorescent indicator was applied to the preparation to image intracellular calcium. A custom-built upright two-photon microscope was used to image the preparation using three dimensional scanning. We were able to image about 1/3 of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we found that outer hair cells demonstrated increased fluorescence compared with surrounding supporting cells. Thus, this methodology can be used to visualize hair cell calcium changes and mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are reduced.

Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved.

Here we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs since they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and bovine serum albumin, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context. PMID:18647170

We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50–500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G{sup *}. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained in force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods.

Full understanding of the physics underlying the striking changes in viscoelasticity, relaxation time, and phase transitions that mesoscopic fluid-like films undergo at solid-liquid interfaces, or under confinement between two sliding solid boundaries, constitutes one of the major challenges in condensed matter physics. Their role in the imaging process of solid substrates by scanning probe microscopy (SPM) is also currently controversial. Aiming at improving the reliability and versatility of instrumentation dedicated to characterize mesoscopic films, a noninvasive whispering-gallery acoustic sensing (WGAS) technique is introduced; its application as feedback control in SPM is also demonstrated. To illustrate its working principle and potential merits, WGAS has been integrated into a SPM that uses a sharp tip attached to an electrically driven 32-kHz piezoelectric tuning fork (TF), the latter also tighten to the operating microscope's frame. Such TF-based SPMs typically monitor the TF's state of motion by electrical means, hence subjected to the effects caused by the inherent capacitance of the device (i.e., electrical resonance differing from the probe's mechanical resonance). Instead, the novelty of WGAS resides in exploiting the already existent microscope's frame as an acoustic cavity (its few centimeter-sized perimeter closely matching the operating acoustic wavelength) where standing-waves (generated by the nanometer-sized oscillations of the TF's tines) are sensitively detected by an acoustic transducer (the latter judiciously placed around the microscope's frame perimeter for attaining maximum detection). This way, WGAS is able to remote monitoring, via acoustic means, the nanometer-sized amplitude motion of the TF's tines. (This remote-detection method resembles the ability to hear faint, but still clear, levels of sound at the galleries of a cathedral, despite the extraordinary distance location of the sound source.) In applications aiming at

Full understanding of the physics underlying the striking changes in viscoelasticity, relaxation time, and phase transitions that mesoscopic fluid-like films undergo at solid-liquid interfaces, or under confinement between two sliding solid boundaries, constitutes one of the major challenges in condensed matter physics. Their role in the imaging process of solid substrates by scanning probe microscopy (SPM) is also currently controversial. Aiming at improving the reliability and versatility of instrumentation dedicated to characterize mesoscopic films, a noninvasive whispering-gallery acoustic sensing (WGAS) technique is introduced; its application as feedback control in SPM is also demonstrated. To illustrate its working principle and potential merits, WGAS has been integrated into a SPM that uses a sharp tip attached to an electrically driven 32-kHz piezoelectric tuning fork (TF), the latter also tighten to the operating microscope's frame. Such TF-based SPMs typically monitor the TF's state of motion by electrical means, hence subjected to the effects caused by the inherent capacitance of the device (i.e., electrical resonance differing from the probe's mechanical resonance). Instead, the novelty of WGAS resides in exploiting the already existent microscope's frame as an acoustic cavity (its few centimeter-sized perimeter closely matching the operating acoustic wavelength) where standing-waves (generated by the nanometer-sized oscillations of the TF's tines) are sensitively detected by an acoustic transducer (the latter judiciously placed around the microscope's frame perimeter for attaining maximum detection). This way, WGAS is able to remote monitoring, via acoustic means, the nanometer-sized amplitude motion of the TF's tines. (This remote-detection method resembles the ability to hear faint, but still clear, levels of sound at the galleries of a cathedral, despite the extraordinary distance location of the sound source.) In applications aiming at

We propose a nanoindentation technique based on atomic force microscopy (AFM) that allows one to deduce both indentation modulus and hardness of viscoelastic materials from the force versus penetration depth dependence, obtained by recording the AFM cantilever deflection as a function of the sample vertical displacement when the tip is pressed against (loading phase) and then removed from (unloading phase) the surface of the sample. Reliable quantitative measurements of both indentation modulus and hardness of the investigated sample are obtained by calibrating the technique through a set of different polymeric samples, used as reference materials, whose mechanical properties have been previously determined by standard indentation tests. By analyzing the dependence of the cantilever deflection versus time, the proposed technique allows one to evaluate and correct the effect of viscoelastic properties of the investigated materials, by adapting a post-experiment data processing procedure well-established for standard depth sensing indentation tests. The technique is described in the case of the measurement of indentation modulus and hardness of a thin film of poly(3,4-ethylenedioxythiophene) doped with poly(4-styrenesulfonate), deposited by chronoamperometry on an indium tin oxide (ITO) substrate. PMID:19674843

Mixed Langmuir monolayers of 10,12-Pentacosadiynoic acid (DA) and amphiphilic hemicyanine (HSP) have been fabricated at the air-water interface. The mixed monolayer has been proved to be completely homogeneous. The DA molecules are arranged in a single monolayer within the mixed Langmuir monolayer, as opposed to the typical trilayer architecture for the pure DA film. Brewster angle microscopy has been used to reveal the mesoscopic structure of the mixed Langmuir monolayer. Flower shape domains with internal anisotropy due the ordered alignment of hemicyanine groups have been observed. Given the absorption features of the hemicyanine groups at the wavelength used in the BAM experiments, the enhancement of reflection provoked by the absorption process leads to the observed anisotropy. The ordering of such groups is promoted by their strong self-aggregation tendency. Under UV irradiation at the air-water interface, polydiacetylene (PDA) has been fabricated. In spite a significant increase in the domains reflectivity has been observed owing to the modification in the mentioned enhanced reflection, the texture of the domains remains equal. The PDA polymer chain therefore grows in the same direction in which the HSP molecules are aligned. This study is expected to enrich the understanding and design of fabrication of PDA at interfaces. PMID:26263495

Magnetic skyrmions are promising candidates as information carriers in logic or storage devices thanks to their robustness, guaranteed by the topological protection, and their nanometric size. Currently, little is known about the influence of parameters such as disorder, defects, or external stimuli on the long-range spatial distribution and temporal evolution of the skyrmion lattice. Here, using a large (7.3×7.3 μm2) single-crystal nanoslice (150 nm thick) of Cu2OSeO3, we image up to 70,000 skyrmions by means of cryo-Lorentz transmission electron microscopy as a function of the applied magnetic field. The emergence of the skyrmion lattice from the helimagnetic phase is monitored, revealing the existence of a glassy skyrmion phase at the phase transition field, where patches of an octagonally distorted skyrmion lattice are also discovered. In the skyrmion phase, dislocations are shown to cause the emergence and switching between domains with different lattice orientations, and the temporal fluctuation of these domains is filmed. These results demonstrate the importance of direct-space and real-time imaging of skyrmion domains for addressing both their long-range topology and stability. PMID:26578765

Magnetic skyrmions are promising candidates as information carriers in logic or storage devices thanks to their robustness, guaranteed by the topological protection, and their nanometric size. Currently, little is known about the influence of parameters such as disorder, defects, or external stimuli on the long-range spatial distribution and temporal evolution of the skyrmion lattice. Here, using a large (7.3 × 7.3 μm(2)) single-crystal nanoslice (150 nm thick) of Cu2OSeO3, we image up to 70,000 skyrmions by means of cryo-Lorentz transmission electron microscopy as a function of the applied magnetic field. The emergence of the skyrmion lattice from the helimagnetic phase is monitored, revealing the existence of a glassy skyrmion phase at the phase transition field, where patches of an octagonally distorted skyrmion lattice are also discovered. In the skyrmion phase, dislocations are shown to cause the emergence and switching between domains with different lattice orientations, and the temporal fluctuation of these domains is filmed. These results demonstrate the importance of direct-space and real-time imaging of skyrmion domains for addressing both their long-range topology and stability. PMID:26578765

The photo-response of semiconductors is usually studied by detecting the photocurrent across source-drain electrodes under light illumination. By integrating the microwave impedance microscopy (MIM) technique with focused-laser stimulation, we are able to perform the real-space photoconductivity mapping of photo-sensitive materials without the need of patterning contact electrodes. Here, we report the MIM results of various thin-film materials, such as In2Se3 nano-sheets and transition metal dichalcogenides (TMD) flakes, illuminated by laser beams of different wavelengths in the ambient condition. With no or below-gap illumination, the samples were highly resistive, as indicated by the low MIM signals. The MIM contrast emerges under above-gap light and increases as increasing laser intensity, which clearly demonstrates the local imaging of photoconductivity rather than the transport photocurrent. Interestingly, clear domain structures with mesoscopic length scales were seen in the data due to the coexistence of multiple phases in In2Se3. The unique combination of MIM and laser stimulation thus provides a new direction to explore the microscopic origin of various light-driven phenomena in complex systems. We gratefully acknowledge financial support from NSF.

FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic hypothesis', a series of proton pumps generate the necessary pH difference plus an electric potential across the bacterial plasma membrane. These proton pumps are redox-coupled membrane enzymes which are possibly organized in supercomplexes, as shown for the related enzymes in the mitochondrial inner membrane. We report diffusion measurements of single fluorescent FoF1-ATP synthases in living E. coli by localization microscopy and single enzyme tracking to distinguish a monomeric enzyme from a supercomplex-associated form in the bacterial membrane. For quantitative mean square displacement (MSD) analysis, the limited size of the observation area in the membrane with a significant membrane curvature had to be considered. The E. coli cells had a diameter of about 500 nm and a length of about 2 to 3 μm. Because the surface coordinate system yielded different localization precision, we applied a sliding observation window approach to obtain the diffusion coefficient D = 0.072 μm2/s of FoF1-ATP synthase in living E. coli cells.

Scanning ion conductance microscopy (SICM) is an increasingly useful nanotechnology tool for non-contact, high resolution imaging of live biological specimens such as cellular membranes. In particular, approach-retract-scanning (ARS) mode enables fast probing of delicate biological structures by rapid and repeated approach/retraction of a nano-pipette tip. For optimal performance, accurate control of the tip position is a critical issue. Herein, we present a novel closed-loop control strategy for the ARS mode that achieves higher operating speeds with increased stability. The algorithm differs from that of most conventional (i.e., constant velocity) approach schemes as it includes a deceleration phase near the sample surface, which is intended to minimize the possibility of contact with the surface. Analysis of the ion current and tip position demonstrates that the new mode is able to operate at approach speeds of up to 250 μm s-1. As a result of the improved stability, SICM imaging with the new approach scheme enables significantly improved, high resolution imaging of subtle features of fixed and live cells (e.g., filamentous structures & membrane edges). Taken together, the results suggest that optimization of the tip approach speed can substantially improve SICM imaging performance, further enabling SICM to become widely adopted as a general and versatile research tool for biological studies at the nanoscale level.

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral–pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology. PMID:26940983

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral-pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology. PMID:26940983

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of “Index of Plasticity” (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus ((H)/(E*)).

Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells. PMID:26034312

There is increasing evidence showing that cytosolic lipid droplets, present in all eukaryotic cells, play a key role in many cellular functions. Yet their composition at the individual droplet level and how it evolves over time in living cells is essentially unknown due to the lack of suitable quantitative nondestructive measurement techniques. In this work, we demonstrate the ability of label-free hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy, together with a quantitative image analysis algorithm developed by us, to quantify the lipid type and content in vol/vol concentration units of individual lipid droplets in living human adipose-derived stem cells during differentiation over 9 days in media supplemented with different fatty acids. Specifically, we investigated the addition of the polyunsaturated linoleic and alpha-linolenic fatty acids into the normal differentiation medium (mostly containing monounsaturated fatty acids). We observe a heterogeneous uptake which is droplet-size dependent, time dependent, and lipid dependent. Cells grown in linoleic-acid-supplemented medium show the largest distribution of lipid content across different droplets at all times during differentiation. When analyzing the average lipid content, we find that adding linoleic or alpha-linolenic fatty acids at day 0 results in uptake of the new lipid components with an exponential time constant of 22 ± 2 h. Conversely, switching lipids at day 3 results in an exponential time constant of 60 ± 5 h. These are unprecedented findings, exemplifying that the quantitative imaging method demonstrated here could open a radically new way of studying and understanding cytosolic lipid droplets in living cells. PMID:26937957

Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 μM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented. PMID:26826690

Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called "z-stacks". However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell

Thin films are attracting more and more attention in both the industrial and scientific communities. Many applications of thin films have been developed in industry. By using various growth methods, thin films can be used in optics, microelectronic devices, magnetic recording media, and as protective coatings. In order to improve existing applications and to find new ones, it is essential to understand what makes them so useful in applications and what factors affect their properties. Therefore, an understanding of film growth processes is necessary. Scientifically, many fundamental interactions, such as the interaction between the atoms that comprise the film and substrate, or the interaction between film atoms, are of great interest to surface scientists; studies of these interactions can provide dramatic insights into the nature of thin films and therefore, can further drive technology forward. In every application, the film structures, including morphology and microstructure, and adhesion between film and substrate are critical to the film`s properties and therefore its performance. Studies of the mechanisms that control film morphology, microstructure and adhesion thus are important. Film growth kinetics can provide important information regarding the film structure and adhesion. Film growth is an atomistic process. The chemistry and physics of the system can be better understood if the information provided is at an atomic level.

Compared the effectiveness of live versus videotape delivery of a behavioral treatment package for enuresis. Outcome was superior for the live delivery. Pretreatment measures of family and child psychosocial adjustment failed to predict treatment response. Film delivery resulted in higher confidence in children of their parents, but lower…

We observed thermomagnetically recorded domains of various sizes with magnetic transmission X-ray microscopy (M-TXM) in order to clarify the recording characteristics. The domains were recorded on TbFeCo films by laser-pumped magnetic field modulation (LP-MFM) using a 635 nm laser diode. Typical images of magnetic domains in TbFeCo films were taken at the Fe L3-edge, and it was confirmed that the crescent-shaped domains could be recorded with high quality for mark lengths down to 100 nm.

In-situ bright field transmission electron microscopy (TEM) nanomechanical tensile testing and in-situ automated crystallographic orientation mapping in TEM were combined to unravel the elementary mechanisms controlling the plasticity of ultrafine grained Aluminum freestanding thin films. The characterizations demonstrate that deformation proceeds with a transition from grain rotation to intragranular dislocation glide and starvation plasticity mechanism at about 1% deformation. The grain rotation is not affected by the character of the grain boundaries. No grain growth or twinning is detected.

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility. PMID:16798949

Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo-thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 μM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake. PMID:21935123

Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo-thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 µM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake. PMID:21935123

All films were deposited by resistive heated evaporation in an oil diffusion pumped vacuum system (ultimate approx. equal to 0.0000001 torr). The growth from nuclei to a continuous film is highly dependent on the deposition parameters, evaporation rate as well as substrate material and substrate temperature. The growth stages of a film and the dependence of grain size on various deposition and annealing parameters are shown. Resistivity measurements were taken on thin films to observe size effects.

A recently developed technique, electrochemical strain microscopy (ESM), utilizes the strong coupling between ionic current and anisotropic volumetric chemical expansion of lithium-ion electrode materials to dynamically probe the sub-one-hundred? nm inter-facial kinetic intercalation properties. A numerical technique based on the finite element method was developed to analyze the underlying physics that govern the ESM signal generation and establish relations to battery performance. The performed analysis demonstrates that the diffusion path within a thin film is tortuous and the extent of lithium diffusion into the electrode is dependent on the SPM-tip-imposed overpotential frequency. The detected surface actuation gives rise to the development of an electromechanical hysteresis loop whose shape is dependent on grain size and overpotential frequency. Shape and tilting angle of the loop are classified into low and high frequency regimes, separated by a transition frequency which is also a function of lithium diffusivity and grain size, f{sub T} = D//{sup 2}. Research shows that the crystallographic orientation of the surface actuated grain has a significant impact on the shape of the loop. The polycrystalline crystallographic orientation of the grains induces a diffusion path network in the electrode which impacts on the mechanical reliability of the battery. Simulations demonstrate that continuous battery cycling results in a cumulative capacity loss as a result of the hysteric non-reversible lithium intercalation. Furthermore, results suggest that ESM has the capability to infer the local out-of-plane lithium diffusivity and the out-of-plane contribution to Vegard tensor.

A recently developed technique, electrochemical strain microscopy (ESM), utilizes the strong coupling between ionic current and anisotropic volumetric chemical expansion of lithium-ion electrode materials to dynamically probe the sub-one-hundred? nm inter-facial kinetic intercalation properties. A numerical technique based on the finite element method was developed to analyze the underlying physics that govern the ESM signal generation and establish relations to battery performance. The performed analysis demonstrates that the diffusion path within a thin film is tortuous and the extent of lithium diffusion into the electrode is dependent on the SPM-tip-imposed overpotential frequency. The detected surface actuation gives rise to the development of an electromechanical hysteresis loop whose shape is dependent on grain size and overpotential frequency. Shape and tilting angle of the loop are classified into low and high frequency regimes, separated by a transition frequency which is also a function of lithium diffusivity and grain size, fT = D/l₂. Research shows that the crystallographic orientation of the surface actuated grain has a significant impact on the shape of the loop. The polycrystalline crystallographic orientation of the grains induces a diffusion path network in the electrode which impacts on the mechanical reliability of the battery. Simulations demonstrate that continuous battery cycling results in a cumulative capacity loss as a result of the hysteric non-reversible lithium intercalation. Furthermore, results suggest that ESM has the capability to infer the local out-of-plane lithium diffusivity and the out-of-plane contribution to Vegard tensor.

Bi₅FeTi₃O₁₅ (BFTO) based Aurivillius ferroelectric thin films were fabricated on strontium ruthanate coated amorphous fused silica substrates using pulsed laser deposition technique. Optical, ferroelectric, and piezoresponse properties of these thin films were investigated. The estimated refractive index (n) and extinction coefficient (k) for these films were in the range from 2.40 to 2.59 and 0.012 to 0.19, respectively. The bandgap of the BFTO thin layers was estimated to be 2.88 eV. Domain switching and hysteresis loops of BFTO films were studied utilizing piezoresponse force microscopy (PFM). The measured apparent polarization (P{sub r}) and coercive field (E{sub c}) for the samples were 20 μC/cm² and 250 kV/cm, respectively. The amplitude and phase hysteresis curves obtained from PFM characterization reveal that these films can be switched below 5 V. These results suggest that BFTO in thin film form is a promising material for photo ferroelectric and optoelectronic devices applications.

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60-250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of D = |0.0054±0.0064| µm(2)/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions. PMID:22470534

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60–250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of = |0.0054±0.0064| µm2/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions. PMID:22470534

Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

The propagation of domain walls in a ferromagnetic film is largely determined by domain-wall pinning at defects in the material. In this article, we map the effective potential landscape for domain-wall pinning in permalloy films by raster scanning a single ferromagnetic vortex and monitoring the hysteretic vortex displacement vs applied magnetic field. The measurement is carried out using a differential magneto-optical microscopy technique which yields spatial sensitivity of approximately 10 nm. We present a simple algorithm for extracting an effective pinning potential from the measurement of vortex displacement vs applied field. The resulting maps of the pinning potential reveal distinct types of pinning sites, which we attribute to quasi-zero-, one-, and two-dimensional defects in the permalloy film.

Atomic force acoustic microscopy (AFAM) is a useful nondestructive technique for measurement of local elastic modulus of materials at nano-scale spatial resolution by measuring the contact resonance spectra for higher order modes of the AFM cantilever. The elastic modulus of RF sputtered HfO{sub 2} thin film has been measured quantitatively, using reference approach in which measurements are performed on the test and reference samples. Using AFAM, the measured elastic modulus of the HfO{sub 2} thin film is 223±27 GPa, which is in agreement with the literature value of 220±40 GPa for atomic layer deposited HfO{sub 2} thin film using nanoindentation technique.

Ultra-thin Bi films show exotic electronic structure and novel quantum effects, especially the widely studied Bi (111) film. Using reflection high-energy electron diffraction and scanning tunneling microscopy, we studied the structure and morphology evolution of Bi (111) thin films grown on Bi{sub 2}Se{sub 3}. A strongly compressed, but quickly released in-plane lattice of Bi (111) is found in the first three bilayers. The first bilayer of Bi shows a fractal growth mode with flat surface, while the second and third bilayer show a periodic buckling due to the strong compression of the in-plane lattice. The lattice slowly changes to its bulk value with further deposition of Bi.

The University of Nebraska is currently evaluating Low Earth Orbit (LEO) simulation techniques as well as a variety of thin film protective coatings to withstand atomic oxygen (AO) degradation. Both oxygen plasma ashers and an electron cyclotron resonance (ECR) source are being used for LEO simulation. Thin film coatings are characterized by optical techniques including Variable Angle Spectroscopic Ellipsometry, Optical spectrophotometry, and laser light scatterometry. Atomic Force Microscopy (AFM) is also used to characterize surface morphology. Results on diamondlike carbon (DLC) films show that DLC degrades with simulated AO exposure at a rate comparable to Kapton polyimide. Since DLC is not as susceptible to environmental factors such as moisture absorption, it could potentially provide more accurate measurements of AO fluence on short space flights.

We report detailed studies of the dynamics of the mitochondrial reticulum in live quiescent cells using two independent experimental techniques: patterned fluorescence correlation spectroscopy (PFCS) and digital video fluorescence microscopy (DVFM). The systems studied consist of osteosarcoma cells stained with Mitotracker Orange and JC-1. In principle, DVFM and PFCS yield identical information about the system, albeit by different analyses. When both methods are used to study the same system it is possible to directly compare measurements of preaveraged statistical dynamical quantities with their microscopic counterparts. This approach allows the underlying mechanism of the observed rates to be determined. Our results indicate that the dynamics of the reticulum structure is composed of two independent contributions, each important on very different time and length scales. During short time intervals ( ~ 1 sec), local regions of the reticulum primarily undergo constrained thermally activated motion. During long time intervals ( ~ 60 sec), we observe local regions of the reticulum to undergo long-range "jump" motion that is associated with the action of cytoskeletal filaments. The frequency of the jumps depend on the physiological state of the cells, however the average jump distance ( ~ 0.6 mm) is unaffected by metabolic activity.

Actomyosin stress fibers, one of the main components of the cell's cytoskeleton, provide mechanical stability to adherent cells by applying and transmitting tensile forces onto the extracellular matrix (ECM) at the sites of cell-ECM adhesion. While it is widely accepted that changes in spatial and temporal distribution of stress fibers affect the cell's mechanical properties, there is no quantitative knowledge on how stress fiber amount and organization directly modulate cell stiffness. We address this key open question by combining atomic force microscopy with simultaneous fluorescence imaging of living cells, and combine for the first time reliable quantitative parameters obtained from both techniques. We show that the amount of myosin and (to a lesser extent) actin assembled in stress fibers directly modulates cell stiffness in adherent mouse fibroblasts (NIH3T3). In addition, the spatial distribution of stress fibers has a second-order modulatory effect. In particular, the presence of either fibers located in the cell periphery, aligned fibers or thicker fibers gives rise to reinforced cell stiffness. Our results provide basic and significant information that will help design optimal protocols to regulate the mechanical properties of adherent cells via pharmacological interventions that alter stress fiber assembly or via micropatterning techniques that restrict stress fiber spatial organization. PMID:26206449

Soft magnetic alloys play a significant role for magnetic recording applications and highly sensitivity magnetic field sensors. In order to sustain the magnetic areal density growth, development of new synthesis techniques and materials is necessary. In this work, the effect of oxygen incorporation during electrodeposition of CoFe alloys on magnetic properties, magnetoresistance and structural properties has been studied. Understanding the magnetic properties often required knowledge of oxygen distribution and structural properties of the grown films. Transmission electron microscopy (TEM) was a powerful tool in this study to correlate the oxygen-distribution nanostructure to the magnetic properties of deposited films. Off-axis electron holography in TEM was used to measure magnetic domain wall width in the deposited films. Elemental depth profiles of Fe, Co, O were investigated by secondary ion mass spectroscopy (SIMS). Magnetic properties have been determined by superconducting quantum interference device (SQUID) measurements. Oxygen content in the CoFe deposited films was controlled by electrolyte composition. Films were deposited on Si 100 substrates and on other substrates such as Cu and Al. However, a good film quality was achieved on Si substrate. Electron energy loss and x-ray spectroscopies showed that the low oxygen films contained intragranular Fe2+ oxide (FeO) particles and that the high oxygen films contained intergranular Fe 3+ (Fe2O3) along grain boundaries. The films with oxide present at the grain boundary had significantly increased coercivity, magnetoresistance and reduced saturation magnetization relative to the lower oxygen content films with intragranular oxide. The differences in magnetic properties between low oxygen and high oxygen concentration films were attributed to stronger mobile domain wall interactions with the grain boundary oxide layers. The very high magnetoresistance values were achieved for magnetic devices with nanocontact

When a liquid droplet impacts on a solid surface, it not only deforms substantially but also an air film develops between the droplet and the surface. This thin air film—as well as other transparent films—can be characterized by reflection interference microscopy. Even for weakly reflecting interfaces, relative thickness variations of the order of tens of nanometers are easily detected, yet the absolute thickness is generally known only up to an additive constant which is a multiple of half of the wavelength. Here, we present an optical setup for measuring the absolute film thickness and its spatial and temporal behavior using a combination of a standard Hg lamp, an optical microscope, and three synchronized high-speed cameras to detect conventional side-view images as well as interferometric bottom view images at two different wavelengths. The combination of a dual wavelength approach with the finite coherence length set by the broad bandwidth of the optical filters allows for measuring the absolute thickness of transient air films with a spatial resolution better than 30 nm at 50 μs time resolution with a maximum detectable film thickness of approximately 8 μm. This technique will be exploited in Part II to characterize the air film evolution during low velocity droplet impacts.

Reflection high energy electron diffraction (RHEED) is commonly used as an in situ analysis tool for layer-by-layer thin-film growth. Atomic force microscopy is an equally common ex situ tool for analysis of the film surface, providing visual evidence of the surface morphology. During growth, the RHEED intensity oscillates as the film surface changes in roughness. It is often assumed that the maxima of the RHEED oscillations signify a complete layer, however, the oscillations in oxide systems can be misleading. Thus, using only the RHEED maxima is insufficient. X-ray reflectivity can also be used to analyze growth, as the intensity oscillates in phase with the smoothness of the surface. Using x-ray reflectivity to determine the thin film layer deposition, we grew three films where the x-ray and RHEED oscillations were nearly exactly out of phase and halted deposition at different points in the growth. Pre-growth and post-growth AFM images emphasize the fact that the maxima in RHEED are not a justification for determining layer completion. Work conducted at the Cornell High Energy Synchrotron Source (CHESS) supported by NSF Awards DMR-1332208 and DMR-0936384 and the Cornell Center for Materials Research Shared Facilities are supported through DMR-1120296.

Ferromagnetic domain patterns and three-dimensional domain-wall configurations in thin CoCrPt films with perpendicular magnetic anisotropy were studied in detail by combining magnetic force microscopy and polarized neutron reflectometry with micromagnetic simulations. With the first method, lateral dimension of domains with alternative magnetization directions normal to the surface and separated by domain walls in 20-nm-thick CoCrPt films were determined in good agreement with micromagnetic simulations. Quantitative analysis of data on reflectometry shows that domain walls consist of a Bloch wall in the center of the thin film, which is gradually transformed into a pair of Néel caps at the surfaces. The width and in-depth thickness of the Bloch wall element, transition region, and Néel caps are found consistent with micromagnetic calculations. A complex structure of domain walls serves to compromise a competition between exchange interactions, keeping spins parallel, magnetic anisotropy orienting magnetization normal to the surface, and demagnetizing fields, promoting in-plane magnetization. It is shown that the result of such competition strongly depends on the film thickness, and in the thinner CoCrPt film (10 nm thick), simple Bloch walls separate domains. Their lateral dimensions estimated from neutron scattering experiments agree with micromagnetic simulations.

Between 1932 and 1963 University of Pittsburgh anatomist Davenport Hooker, Ph.D., performed and filmed noninvasive studies of reflexive movement on more than 150 surgically aborted human fetuses. The resulting imagery and information would contribute substantially to new visual and biomedical conceptions of fetuses as baby-like, autonomous human entities that emerged in the 1960s and 1970s. Hooker's methods, though broadly conforming to contemporary research practices and views of fetuses, would not have been feasible later. But while Hooker and the 1930s medical and general public viewed live fetuses as acceptable materials for nontherapeutic research, they also shared a regard for fetuses as developing humans with some degree of social value. Hooker's research and the various reactions to his work demonstrate the varied and changing perspectives on fetuses and fetal experimentation, and the influence those views can have on biomedical research. PMID:24769805

Thin films of the quasi one-dimensional organic conductor TTF-TCNQ grown on KCl (001) substrates by Chemical Vapor Deposition has been analyzed with Atomic Force Microscopy. The films are polycrystalline, composed of microcrystals with rectangular shape with the c∗ crystallographic axis perpendicular to the substrate. The stepped surface morphology of the microcrystals has been studied. The growth of the films is strongly dominated by an oriented nucleation and the one-dimensional nature of the compound.

The applicability of a femtosecond KrF laser in absorbing film-assisted, laser-induced forward transfer of living cells was studied. The absorbing materials were 50-nm-thick metal films and biomaterials (gelatine, Matrigel, each 50 μm thick, and polyhydroxybutyrate, 2 μm). The used cell types were human neuroblastoma, chronic myeloid leukemia, and osteogenic sarcoma cell lines, and primary astroglial rat cells. Pulses of a 500-fs KrF excimer laser focused onto the absorbing layer in a 250-μm diameter spot with 225 mJ/cm2 fluence were used to transfer the cells to the acceptor plate placed at 0.6 mm distance, which was a glass slide either pure or covered with biomaterials. While the low-absorptivity biomaterial absorbing layers proved to be ineffective in transfer of cells, when applied on the surface of acceptor plate, the wet gelatine and Matrigel layers successfully ameliorated the impact of the cells, which otherwise did not survive the arrival onto a hard surface. The best short- and long-term survival rate was between 65% and 70% for neuroblastoma and astroglial cells. The long-term survival of the transferred osteosarcoma cells was low, while the myeloid leukemia cells did not tolerate the procedure under the applied experimental conditions.

We assess scanning electron microscopy (SEM) and Auger electron spectroscopy (AES) for thickness measurements on few-layer hexagonal boron nitride (h-BN), the layered dielectric of choice for integration with graphene and other two-dimensional materials. Observations on h-BN islands with large, atomically flat terraces show that the secondary electron intensity in SEM reflects monolayer height changes in films up to least 10 atomic layers thickness. From a quantitative analysis of AES data, the energy-dependent electron escape depth in h-BN films is deduced. The results show that AES is suitable for absolute thickness measurements of few-layer h-BN of 1 to 6 layers.

X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue—magnetic X-ray tomography—is yet to be developed. Here we demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. The 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. Using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomena between windings of azimuthally and radially magnetized tubular objects. The present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape. PMID:26139445

X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue—magnetic X-ray tomography—is yet to be developed. We demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. In the 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. By using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomenamore » between windings of azimuthally and radially magnetized tubular objects. In conclusion, the present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape.« less

X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue—magnetic X-ray tomography—is yet to be developed. We demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. In the 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. By using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomena between windings of azimuthally and radially magnetized tubular objects. In conclusion, the present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape.

X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue--magnetic X-ray tomography--is yet to be developed. Here we demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. The 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. Using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomena between windings of azimuthally and radially magnetized tubular objects. The present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape. PMID:26139445

X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue--magnetic X-ray tomography--is yet to be developed. Here we demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. The 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. Using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomena between windings of azimuthally and radially magnetized tubular objects. The present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape.

Scanning tunneling microscopy (STM) is used to study the basic laws of growth of ultrathin epitaxial CoSi2(111) films with Co coverages up to 4 ML formed upon sequential deposition of Co and Si atoms taken in a stoichiometric ratio onto the Co-Si(111) surface at room temperature and subsequent annealing at 600-700°C. When the coverage of Co atoms is lower than ~2.7 ML, flat CoSi2 islands up to ~3 nm high with surface structure 2 × 2 or 1 × 1 grow. It is shown that continuous epitaxial CoSi2 films containing 3-4 triple Si-Co-Si layers grow provided precise control of deposition. CoSi2 films can contain inclusions of the local regions with (2 × 1)Si reconstruction. At a temperature above 700°C, a multilevel CoSi2 film with pinholes grows because of vertical growth caused by the difference between the free energies of the CoSi2(111) and Si(111) surfaces. According to theoretical calculations, structures of A or B type with a coordination number of 8 of Co atoms are most favorable for the CoSi2(111)2 × 2 interface.

Maskless patterning of biocompatible Ta2O5/Pt/glass sensor chips can be realized by ultra-short laser pulse ablation. At a fluence of 0.2 J/cm2, the thin Ta2O5 film is selectively lifted-off by indirectly-induced ablation at laser wavelenghts where the Ta2O5 is transparent and the Pt absorbing. This enables precise and very fast structuring. Here, 660 fs laser pulses at a center wavelength of 1053 nm are applied. The driving physical effects of this ablation mechanism are revealed by pump-probe microscopy. This technique allows the observation of the whole ablation process ranging temporally from femtoseconds to microseconds. An ultrafast heat-expansion in the absorbing Pt, initiating a shock-wave to the Ta2O5 within the first 10 ps, bulges the Ta2O5 film after some nanoseconds. Bulging velocities of 750 m/s are determined corresponding to an extreme acceleration of about 1010 g. Exceeding the stress limit in the Ta2O5 causes film disintegration after 50 ns. A model, describing essential reaction steps, is developed. This model is also applicable to other industrial important layer systems, where thin transparent films have to be removed.

In this report, conditions for attaining high resolution in scanning electron microscopy with soft biological specimens are described using the currently available high resolution scanning electron microscopes in emission mode of low energy electrons (secondary and charging electrons). Retinal rod outer segments, red blood cells, intestinal mucosa, and ferritin molecules were all used as biological test specimens. From uncoated specimens a new source of signal, referred to as a discharge signal, can provide a high yield of low energy electrons from an excitation area approximately the size of the beam's cross section. Additionally, under these conditions sufficient topographic contrast can be achieved by applying ultra thin metal coatins. A 0.5 nm thick gold film is found sufficient for generating the total signal, whereas increased coating thickness causes additional topographic background signal. However, a 2.0 nm film is needed for imaging surface details with the present instrument. Ultra thin, even, and grainless tantalum films have been found effective in eliminating the charging artifacts caused by external fields, and the decoration artifacts caused by crystal growth as seen in gold films. To improve, in high magnification work on ultra thin coated specimen, signal-to-noise ratio, methods for obtaining saturation of the signal with discharge electrons are shown. The necessity of confirming the information obtained in SEM by independent techniques (TEM of stereo-replicas or ultra thin sections) is discussed. PMID:392703

We demonstrate three-level data storage in amorphous Ge2Sb2Te5 (GST) thin film by conductive atomic force microscopy (C-AFM). Due to the high resolution and current sensitivity of AFM, the electrical properties of GST are investigated in the nanoscale. By applying an electric field between an AFM probe tip and the GST surface, well-resolved threshold switching and memory switching are obtained successively in a current-voltage sweeping. Correspondingly, three states with high, intermediate and low resistances, which are assigned data values ‘0’, ‘1’ and ‘2’ respectively, are observed in an IV-spectrum. The electrical resistance of GST thin film decreases by over two orders of magnitude in both switching processes, which provides a clear contrast to distinguish the three logical states. We also discuss the threshold electrical field of threshold switching in the amorphous GST thin film. Nanoscale conductive marks in the amorphous ON state and crystalline state are successfully fabricated by applying IV-spectra with different voltage ranges on the GST thin films.

We demonstrate three-level data storage in amorphous Ge2Sb2Te5 (GST) thin film by conductive atomic force microscopy (C-AFM). Due to the high resolution and current sensitivity of AFM, the electrical properties of GST are investigated in the nanoscale. By applying an electric field between an AFM probe tip and the GST surface, well-resolved threshold switching and memory switching are obtained successively in a current-voltage sweeping. Correspondingly, three states with high, intermediate and low resistances, which are assigned data values '0', '1' and '2' respectively, are observed in an IV-spectrum. The electrical resistance of GST thin film decreases by over two orders of magnitude in both switching processes, which provides a clear contrast to distinguish the three logical states. We also discuss the threshold electrical field of threshold switching in the amorphous GST thin film. Nanoscale conductive marks in the amorphous ON state and crystalline state are successfully fabricated by applying IV-spectra with different voltage ranges on the GST thin films. PMID:26651151

The local surface potential of pin-type hydrogenated amorphous silicon (a-Si:H) thin-film solar cells has been evaluated by Kelvin force microscopy (KFM). We have also estimated the local built-in potential of the solar cells by KFM. In the surface morphology image of the solar cells, large convex grains related to the textured structure of the substrate were found. The surface potential distribution related to the surface morphology was observed in the solar cells. A similar surface potential distribution was also found in an n-type hydrogenated microcrystalline Si (µc-Si:H) film. The surface potential of the solar cells was not the same as that of the n-type film. The difference in average surface potential between the n-type hydrogenated microcrystalline Si (µc-Si:H) film and the solar cells increased with increasing built-in potential. The difference in local surface potential on large convex grains was smaller than that in the region between the large convex grains.

We investigated the surface work function (WS) and its spatial distribution for epitaxial VO2/TiO2 thin films using Kelvin probe force microscopy (KPFM). Nearly grain-boundary-free samples allowed observation of metallic and insulating domains with distinct WS values, throughout the metal–insulator transition. The metallic fraction, estimated from WS maps, describes the evolution of the resistance based on a two-dimensional percolation model. The KPFM measurements also revealed the fractal nature of the domain configuration. PMID:25982229

CdS films have been deposited by solution growth on SnO{sub 2} and glass substrates. Nucleation on SnO{sub 2} occurs at early deposition times, and complete conformal coverage is observed at low thickness values. The average grain size of the CdS films is established at these early times. In films deposited on glass substrates, nucleation is slower and occurs through 3-dimensional islands that increase in size and number as deposition proceeds. Optical measurements show that the bandgap values of CdS films deposited on SnO{sub 2} depend mainly on substrate structure. Hydrogen heat treatment does not affect the surface morphology of the samples, but decreases bandgap values.

An extremely small, conically shaped Pt microelectrode tip (with a radius of 30 nanometers) and the precise positioning capabilities of the scanning electrochemical microscope were used to penetrate a thin (200 nanometers) polymer film and obtain directly the standard potential and kinetic parameters of an electrode reaction within the film. The thickness of the film was determined while it was immersed in and swollen by an electrolyte solution. The film studied was the perfluorosulfonate Nafion containing Os(bpy)(3)(2+) (bpy, 2,2'-bipyridine) cast on an indium tin oxide surface. The steady-state response at the ultramicroelectrode allowed direct determination of the rate constant for heterogeneous electron transfer K(o) and the diffusion coefficient D without complications caused by transport in the liquid phase, charge exchange at the liquid-polymer interface, and resistive drop. PMID:17832832

The photophysics of a covalently linked perylenediimide–diketopyrrolopyrrole–perylenediimide acceptor–donor–acceptor molecule (PDI–DPP–PDI, 1) were investigated and found to be markedly different in solution versus in unannealed and solvent annealed films. Photoexcitation of 1 in toluene results in quantitative charge separation in τ = 3.1 ± 0.2 ps, with charge recombination in τ = 340 ± 10 ps, while in unannealed/disordered films of 1, charge separation occurs in τ < 250 fs, while charge recombination displays a multiexponential decay in ~6 ns. The absence of long-lived, charge separation in the disordered film suggests that few free charge carriers are generated. In contrast, uponmore » CH₂Cl₂ vapor annealing films of 1, grazing-incidence X-ray scattering shows that the molecules form a more ordered structure. Photoexcitation of the ordered films results in initial formation of a spin-correlated radical ion pair (electron–hole pair) as indicated by magnetic field effects on the formation of free charge carriers which live for ~4 μs. This result has significant implications for the design of organic solar cells based on covalent donor–acceptor systems and shows that long-lived, charge-separated states can be achieved by controlling intramolecular charge separation dynamics in well-ordered systems.« less

The photophysics of a covalently linked perylenediimide–diketopyrrolopyrrole–perylenediimide acceptor–donor–acceptor molecule (PDI–DPP–PDI, 1) were investigated and found to be markedly different in solution versus in unannealed and solvent annealed films. Photoexcitation of 1 in toluene results in quantitative charge separation in τ = 3.1 ± 0.2 ps, with charge recombination in τ = 340 ± 10 ps, while in unannealed/disordered films of 1, charge separation occurs in τ < 250 fs, while charge recombination displays a multiexponential decay in ~6 ns. The absence of long-lived, charge separation in the disordered film suggests that few free charge carriers are generated. In contrast, upon CH₂Cl₂ vapor annealing films of 1, grazing-incidence X-ray scattering shows that the molecules form a more ordered structure. Photoexcitation of the ordered films results in initial formation of a spin-correlated radical ion pair (electron–hole pair) as indicated by magnetic field effects on the formation of free charge carriers which live for ~4 μs. This result has significant implications for the design of organic solar cells based on covalent donor–acceptor systems and shows that long-lived, charge-separated states can be achieved by controlling intramolecular charge separation dynamics in well-ordered systems.

With power conversion efficiencies on the rise, organic photovoltaics (OPVs) hold promise as a next-generation thin-film solar technology. However, both device performance and stability are inextricably linked to local film structure. Methods capable of probing nanoscale electronic properties as a function of film structure are thus a crucial component of the rational design of efficient and robust devices. This dissertation describes the use of three scanning probe methods for studying local charge generation and photodegradation in polymer/fullerene solar cells. First, we show that time-resolved electrostatic force microscopy (trEFM) is capable of resolving local photocurrent from sub-bandgap excitation down to attoampere level currents, a result unattainable by traditional contact-mode methods. We find that the local charging rates measured with trEFM are proportional to external quantum efficiency (EQE) measurements made on completed devices, making trEFM images equivalent to local EQE maps across the entire solar spectrum. For both phase-segregated and well-mixed MDMO-PPV:PCBM film morphologies, we show that the local distribution of photocurrent is invariant to excitation wavelength, providing local evidence for the controversial result that the probability of generating separated charge carriers does not depend on whether excitons are formed at the singlet state or charge transfer state. Next, we describe how local dissipation imaging can be performed with commercially-available frequency-modulated electrostatic force microscopy (FM-EFM) and show that dissipation maps are highly sensitive to photo-oxidative effects in organic semiconductors. We show that photo-oxidation induced changes in cantilever energy dissipation are proportional to device performance losses. We further develop dissipation imaging by implementing ringdown imaging, which directly measures the quality factor of the cantilever, enabling quantitative dissipation mapping. Using organic

Rh nanoparticles (mean size 10 and 15 nm), prepared by epitaxial growth on NaCl surfaces, were covered with layers of crystalline vanadium oxide (mean thickness 1.5 and 25 nm) by reactive deposition in 10(-2) mbar O2. The 1.5 nm film was further stabilized with a coating layer of 25 nm amorphous alumina. The so-obtained Rh/vanadia films, containing vanadium in the V3+ and V2+ state, were treated in 1 bar O2 at 673 K for 1 h and thereafter reduced in 1 bar H2 at increased temperatures, particularly between 723 and 873 K. The structural and morphological changes were followed by (high-resolution) transmission electron microscopy and selected area diffraction. Oxidation at 673 K transforms the purely vanadia-supported samples into Rh/V2O5, while in the alumina-supported films containing only small amounts of VOx, the formation of topotactic V2O3 is observed. The formation of Rh-V alloys during the subsequent reduction is strongly determined by the intimate contact and the structural and orientational relationship between Rh particles and the surrounding VOx phase. Reduction above 473 K transforms the support into substoichiometric vanadium oxides of composition VO and V2O. Analysis of high-resolution images and diffraction patterns reveals the presence of different alloy phases after reduction with increasing T (from 573 up to 823 K). In the alumina-supported film (low V/Rh ratio) the epitaxial alignment between the Rh particles and the surrounding V2O3 phase apparently favours the primary formation of defined alloys of type V3Rh and VRh3, followed by VRh at higher temperature. On the contrary, mainly V3Rh5 is formed in the purely VOx-supported Rh/films, due to different epitaxial relations in the initial state. Possible pathways of alloy formation are discussed. PMID:16633603

The charge trapping properties of the blend of polystyrene (PS) and a sterically hindered organic semiconductor SFDBAO (spiro[fluorene-9,7-dibenzo[c,h]acridin-5-one]) are investigated by electrostatic and Kelvin probe force microscopy (EFM and KPFM). EFM signals of trapped charge spots injected with controllable tip biases, which are recorded with different dissipation times t, the percent of SFDBAO in blends, and the scanning tip bias, have been measured. By the quantitative analysis, the excellent trapped charge density of PS/SFDBAO blend films for the holes (∼×10(-5) C m(-2)) is much higher than that of the SFDBAO film (∼×10(-6) C m(-2)) and the PS film (∼×10(-7) C m(-2)). However, the trapped charge density of electrons (∼×10(-7) C m(-2)) has the same order magnitude for SFDBAO, PS and the blend films. The results indicate that the blend of PS and SFDBAO enhances the high-density storage and retention abilities of the holes to a larger extent, but the endurance improvement of the electrons is not that obvious. By the KPFM measurement, we further verify the different diffusion rates of the trapped holes and electrons in the PS/SFDBAO blend films, and discuss the possible physical mechanism. The qualitative and quantitative determination of charge trapping properties in this work can be very useful for the characterization of PS/SFDBAO based charge trapping memory devices. PMID:26979556

Gold and copper films (0.2-2 micron thick) are ion plated on very smooth stainless steel 304 and mica surfaces. The deposited films are examined by SEM to identify the morphological growth of defects. Three types of coating defects are distinguished: nodular growth, abnormal or runaway growth, and spits. The potential nucleation sites for defect growth are analyzed to determine the cause of defect formation. It is found that nuclear growth is due to inherent surface microdefects, abnormal or runaway growth is due to external surface inclusions, and spits are due to nonuniform evaporation and ejection of droplets. All these defects have adverse effects on the coatings.

Oxide thin-films have attracted considerable attention for a new generation of spintronics devices, where both electron charge and spin are used to transport information. However, a poor understanding of the local features that mediate magnetization and coupling in these materials has greatly limited their deployment into new information and communication technologies. This thesis describes direct, local measurements of structure-property relationships in ferrous thin-films and La1--xSrxMnO3 (LSMO) / Pb(ZrxTi1--x)O3 (PZT) thin-film heterostructures using spatially-resolved characterization techniques. In the first part of this thesis we explore the properties of ferrous spintronic thin-films. These films serve as a model system to establish a suite of interfacial characterization techniques for subsequent studies. We then study the static behavior of LSMO / PZT devices with polarization set by the underlying substrate. Using transmission electron microscopy and geometric phase analysis we reveal the presence of significant local strain gradients in these films for the first time. Electron energy loss spectroscopy mapping of the LSMO / PZT interface reveals Mn valence changes induced by charge-transfer screening. Bulk magnetometry and polarized neutron reflectometry indicate that these chemical and strain changes are associated with a graded magnetization across the LSMO layer. Density functional theory calculations are presented, which show that strain and charge-transfer screening act locally to suppress magnetization in the LSMO by changing the Mn orbital polarization. In the second half of this thesis, we explore asymmetric screening effects on magnetization LSMO / PZT composites. We find that the local ferroelectric polarization can vary widely and that this may be responsible for reduced charge-transfer effects, as well as magnetic phase gradients at interfaces. From this information and electron energy loss spectroscopy, we construct a map of the magnetic

Charge carrier diffusion coefficient and length are important physical parameters for semiconducting materials. Long-range carrier diffusion in perovskite thin films has led to remarkable solar cell efficiencies; however, spatial and temporal mechanisms of charge transport remain unclear. Here we present a direct measurement of carrier transport in space and in time by mapping carrier density with simultaneous ultrafast time resolution and ∼50-nm spatial precision in perovskite thin films using transient absorption microscopy. These results directly visualize long-range carrier transport of ∼220 nm in 2 ns for solution-processed polycrystalline CH3NH3PbI3 thin films. Variations of the carrier diffusion coefficient at the μm length scale have been observed with values ranging between 0.05 and 0.08 cm2 s−1. The spatially and temporally resolved measurements reported here underscore the importance of the local morphology and establish an important first step towards discerning the underlying transport properties of perovskite materials. PMID:26101051

Environmental stress cracking of polymeric materials in the presence of solvents is a well-known phenomenon in which a stressed polymer exposed to solvents will exhibit premature crazing and eventual failure. Here we examine the solvent-polymer interaction between poly(ethylene)terepthalate (PET) films prepared by a spin coating technique and the solvents including water, isopropanol, and nitroethane. The interaction was followed using both tapping-mode atomic force microscopy (AFM) and Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR). Using these methods, we hope to gain a better understanding of the effects of solvent interaction at the surface of these polymer films, and of the mechanism of the stress cracking phenomenon. We examine the morphological characteristics of the films as a function of exposure time to the various solvents, by acquiring both in situ AFM images of the polymer in the solvent, and post-immersion imaging of the solvent-exposed polymer in air. We also correlate the AFM images to more quantitative measurements of the degree of polymer crystallinity as measured by the FTIR-ATR technique. By appropriate choice of solvents we can independently examine the effects both of varying the solubility parameter of the solvent component of the polymer-solvent system and of hydrolysis or esterification of the polymer by the solvent.

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure. PMID:27320699

Kelvin-probe force microscopy (KFM) has been widely used to evaluate the localized charge trap states in the organic thin-film transistor (OTFT) channels. However, applicability of the KFM has been limited to the trapped charges whose lifetime is typically longer than several minutes because of the temporal resolution of the KFM. Therefore, it has not long been employed for studying the dynamics of the trapped charges in the OTFTs. Here, we demonstrate a method to visualize the transient distribution of the trapped charge carriers in operating OTFTs. The method allows visualizing the dynamics of the trapped charges during the gate voltage sweeps on a time scale of several hundreds of milliseconds. The experimental results performed on dinaphtho[2,3-b:2',3'-f]thieno[3,2-b]thiophene (DNTT) OTFTs indicate that, immediately after a bias voltage applied to a device was turned off, the primary discharging of the channel region around the electrode edges started and it limited the ejection process of the remaining accumulated charges to the electrodes, resulting in an increased density of long-lived trapped charges in a region distant from the electrodes. The presented results suggest that the method is useful to study the electrical connections at the interface between the DNTT grains and electrodes, or those between the grains.

Utilizing atomic force microscope (AFM) with a diamond tip, we were able to successfully plough nano-constrictions on epitaxially grown YBa2Cu3O(7-x) thin films deposited on MgO substrates. The thickness, width, and length of the obtained constrictions were in the range of a few 100 nm. Furthermore, we managed to produce a new S-type constriction, of which the dimensions are easier to control than for conventional constrictions. PMID:18019174

Human immunodeficiency virus 1 (HIV-1) is a human retrovirus that efficiently, albeit gradually, overruns the immune system. An already infected T lymphocyte can latch onto another T lymphocyte whereby creating a virological synapse (VS); this junction drives viral assembly and transfer to the target cell in batches in an efficient, protective manor. My Ph.D. doctoral thesis focused on studying this transmission mechanism using advanced optical imaging modalities and the fully infectious fluorescent clone HIV Gag-iGFP. T lymphocytes are non-adherent cells (˜10 um thick) and the viral transmission process is fairly dynamic, hence we employed a custom spinning disk confocal microscope that revealed many interesting characteristics of this cooperative event. This methodology has low throughput as cell contact and transfer is at random. Optical tweezers was then added to the microscope to directly initiate cell contact at will. To assess when viral maturation occurs post-transfer, an optical assay based off of Forster resonance energy transfer was developed to monitor maturation. Structured illumination microscopy was further used to image the process at higher resolution and it showed that viral particles are not entering existing degradative compartments. Non-HIV-1 applications of the optical technologies are also reviewed.

Simple ferromagnetic (FM) and antiferromagnetic (AF) materials such as Fe and Cr become paramagnetic when heated above some critical temperature, in what is known as a second-order phase transition. Less usual magnetic transitions are found in the magnetic world, for example a first-order magnetic phase transition from AF to FM with increasing temperature. Equiatomic FeRh has been known to exhibit such a transition for over 50 years, with a transition temperature slightly above room temperature. Interest in this material has been renewed in the recent years due to its potential application for heat-assisted magnetic recording, as well as a test system for fundamental studies of the physics of magnetic phase transitions. Similarly to crystallization, this AF-FM transition is expected to proceed by nucleation of magnetic domains but the features of the first-order hysteretic transition have been difficult to study with macroscopic measurements and very few microscopic studies have been performed. In this work, FeRh thin films were synthesized by magnetron sputtering and structurally and magnetically characterized. A membrane-based heating device was designed to enable temperature-dependent microscopy measurements, providing a thermally uniform and well-controlled sample area. Synchrotron x-ray magnetic microscopy was used to study the temperature-driven AF-FM phase transition in epitaxial FeRh thin films in zero field. Using magnetic microscopy with x-ray magnetic circular dichroism, the different stages of nucleation, growth and coalescence of FM domains were observed across the transition and details of the nucleation were identified. The FM phase nucleates into single domain islands and the width of the transition of the individual nuclei upon heating is sharper than that of the macroscopic transition. Using magnetic microscopy with x-ray magnetic linear dichroism, the evolution of the AF phase was studied. Differences in the morphology of AF and FM phases were

Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies. We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

Chitinases are widely distributed in nature and have wide-ranging pharmaceutical and biotechnological applications. This work highlights a real-time and label-free method to assay Chitinase activity via a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). The chitin substrate was prepared by spincoating a trimethylsilyl chitin solution onto a silica substrate, followed by regeneration to amorphous chitin (RChi). The QCM-D and AFM results clearly showed that the hydrolysis rate of RChi films increased as Chitinase (from Streptomyces griseus) concentrations increased, and the optimal temperature and pH for Chitinase activity were around 37 °C and 6-8, respectively. The Chitinase showed greater activity on chitin substrates, having a high degree of acetylation, than on chitosan substrates, having a low degree of acetylation. PMID:23822524

Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation. PMID:27272931

We have directly observed motion of inorganic nanoparticles during fluid evaporation using a Transmission Electron Microscope. Tracking real-time diffusion of both spherical (5-15 nm) and rod-shaped (5x10 nm) gold nanocrystals in a thin-film of water-15percentglycerol reveals complex movements, such as rolling motions coupled to large-step movements and macroscopic violations of the Stokes-Einstein relation for diffusion. As drying patches form during the final stages of evaporation, particle motion is dominated by the nearby retracting liquid front.

Introducing inorganic nanoparticles into a polymer is known to modify the macroscopic mechanical properties of the material. This is often interpreted by assuming the presence of a polymer layer with different properties at the interface with the particles. There is however little direct information available on the mechanical properties of such an interfacial layer. We have used an Atomic Force Microscope (AFM) as a nano-indenter to probe the mechanical response of thin poly(styrene butadiene) random copolymers deposited on oxidized silicon wafers (model silica surface). Indentations were performed at different approach and retraction speeds at room temperature (polymer in the rubbery state) on films with thicknesses ranging from 40nm to 500nm. Approach and retraction curves obtained at high speeds are characteristic of the indentation of an elastic material with an adhesive tip/polymer contact. At low speeds, the adhesion forces dominate for low applied forces, while the elasticity of the polymer dominates the behaviour at high applied load. This allows us to separate the mechanical response of the polymer film from the tip-polymer adhesion that involves some dissipation taking place close to the contact line between the polymer free surface and the tip.

We study the ferromagnetic metallic domains from the charge-order insulating background at mesoscopic length scale in a Pr0.55Ca0.75Sr0.25MnO3 thin film using a variable temperature microwave impedance microscope (MIM). The metallic state in this compound can be easily induced at a moderate magnetic field as low as 2 T observed by both the transport and MIM. The temperature dependent transport under 1.2 T shows a large hysteresis loop. MIM allows us to observe the formation and melting of metallic domains at different temperatures during the cooling and warming processes. At higher temperatures, the metallic domains first emerge in small isolated filaments along certain crystal axes of the LSAT(110) substrate, suggesting that the local strain plays an important role. Surprisingly, small insulating islands remain in the metallic ground state and persist up to very high magnetic fields, indicating strong pining sites. Lastly, the sizes of the insulating islands at the ground state increase when the film is field cooled at lower speeds, suggesting s glassy order in this compound.

The Scanning Force Microscope has been used to study the lattice and defect structures of multilayers of the unsaturated fatty acid, 12-8- diacetylene (10,12-Pentacosadiynoic Acid) in ambient conditions. Films were prepared by the Langmuir-Blodgett technique on ordinary microscope glass and Indium Tin Oxide coated glass. Lattice structures were deduced from the well resolved molecular images and before polymerization found to be nearly centered rectangular with lattice parameters (0.88 +/- 0.06)nm and (0.51 +/- 0.04)nm. After exposing to UV radiation for polymerization the lattice structure changed to an oblique lattice with lattice parameters (0.466 +/- 0.008)nm and (0.55 +/- 0.01)nm. Molecular level defects such as dislocations and grain boundaries were resolved in these films very clearly. Observation of these kind of defects implies that it is possible to reliably image the real surface molecules under ambient conditions. Polymerization was found to take place in one of the lattice directions and the modulation perpendicular to that direction was more pronounced than along the polymer backbone.

The reliability of ultrathin organic layers as active components for molecular electronic devices depends ultimately on an accurate characterization of the layer morphology and ability to withstand mechanical stresses on the nanoscale. To this end, since the molecular layers need to be electrically decoupled using thick insulating substrates, the use of AFM becomes mandatory. Here, we show how friction force microscopy (FFM) in water allows us to identify the orientation of copper(ii)phthalocyanine (CuPc) molecules previously self-assembled on a dolomite (104) mineral surface in ultra-high vacuum. The molecular features observed in the friction images show that the CuPc molecules are stacked in parallel rows with no preferential orientation with respect to the dolomite lattice, while the stacking features resemble well the single CuPc crystal structure. This proves that the substrate induction is low and makes friction force microscopy in water a suitable alternative to more demanding dynamic AFM techniques in ultra-high vacuum.

The reliability of ultrathin organic layers as active components for molecular electronic devices depends ultimately on an accurate characterization of the layer morphology and ability to withstand mechanical stresses on the nanoscale. To this end, since the molecular layers need to be electrically decoupled using thick insulating substrates, the use of AFM becomes mandatory. Here, we show how friction force microscopy (FFM) in water allows us to identify the orientation of copper(ii)phthalocyanine (CuPc) molecules previously self-assembled on a dolomite (104) mineral surface in ultra-high vacuum. The molecular features observed in the friction images show that the CuPc molecules are stacked in parallel rows with no preferential orientation with respect to the dolomite lattice, while the stacking features resemble well the single CuPc crystal structure. This proves that the substrate induction is low and makes friction force microscopy in water a suitable alternative to more demanding dynamic AFM techniques in ultra-high vacuum. PMID:24932960

The evolution of interface between the epitaxial thin film LiCoO2 (LCO) electrode and liquid electrolyte and inside the LCO film during electrochemical cycling has been analyzed by high resolution scanning transmission electron microscopy. Relaxation of sharp translational domain boundaries with mismatched layers of CoO2 octahedra occurs during cycling and results in formation of continuous CoO2 layers across the boundaries. The original trigonal layered structure of LiCoO2 tends to change into a spinel structure at the electrode/electrolyte interface after significant extraction of Li from LCO. This change is more pronounced at 4.2 V peak of CV, indicating lower stability of the layered LCO structure near its surface after Li is extracted above 60%. The transformed structure is identified to be close to Co3O4, with Co both on tetrahedral and octahedral sites, rather than to LiCo2O4 as it was suggested in earlier publications. Electron energy-loss spectroscopy measurements also show that Co ions oxidation state is reduced to mixed valence state Co(2+)/Co(3+) during the structure changes to spinel rather than oxidized. PMID:26911456

Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Our results allow for improved safety during laser ablation in manufacturing and medical applications.

Resistive Random Access Memory (RRAM) with a structure Au/Ti/TiO2-x/Au demonstrated a clear bipolar resistive switching behavior without the necessity of an initial electroforming process. The titanium oxide (TiO2-x) thin film was deposited by reactive RF magnetron sputtering at room temperature in a controlled oxygen/argon ambient. The high density of oxygen vacancies within the film (induced by the low oxygen content) is an essential component for the formation of conducting filaments and demonstration of DC or nanosecond pulsed resistance switching, but also impose limitations for the conduction behavior of the high resistance state. Conductive Atomic Force Microscopy (C-AFM) was then employed in order to investigate the nanoscale electrical properties of our device. In situ current distribution during the SET process disclosed possible formation of conducting filaments while DC sweeping bias voltage revealed an OFF/ON switching ratio of about 200. We have also demonstrated that by using C-AFM both a low resistance state and a high resistance state can be written by bipolar voltage application imaged by corresponding patterns on the TiO2-x current image, suggesting that oxygen ions movement at the Pt-Ir coated tip/TiO2-x interface plays a critical role in the resistive switching phenomenon and thus correlating the macroscopic characteristics of our device with its microscopic origins. Nanoscale resistance switching is also demonstrated by programming distinct patterns on the device's current image.

Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Our results allow for improved safety during laser ablation in manufacturing and medical applications. PMID:26965073

Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Our results allow for improved safety during laser ablation in manufacturing and medical applications. PMID:26965073

Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combinationmore » of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Ultimately, our results allow for improved safety during laser ablation in manufacturing and medical applications.« less

Resonantly absorbing thin films comprising periodically sub-wavelength structured metal surface, dielectric spacer, and metal ground plane are a topic of current interest with important applications. These structures are frequently described as "metamaterials", where effective permittivity and permeability with dispersion near electric and magnetic resonances allow impedance matching to free space for maximum absorption. In this paper, we compare synchrotron-based infrared spectral microscopy of a single isolated unit cell and a periodic array, and we show that the resonances have little to do with periodicity. Instead, the observed absorption spectra of usual periodically structured thin films are best described as due to standing-wave resonances within each independent unit cell, rather than as due to effective optical constants of a metamaterial. The effect of having arrays of unit cells is mainly to strengthen the absorption by increasing the fill factor, and such arrays need not be periodic. Initial work toward applying the subject absorbers to room-temperature bolometer arrays is presented.

The surface of 3C SiC films grown on 6H SiC substrates has been studied by atomic probe microscopy in air. Atomic-scale images of the 3C SiC surface have been obtained by STM which confirm the 111 line type orientation of the cubic 3C layer grown on the 0001 plane type surface of the hexagonal 6H substrate. The nearest-neighbor atomic spacing for the 3C layer has been measured to be 3.29 +/- 0.2 A, which is within 7 percent of the bulk value. Shallow terraces in the 3C layer have been observed by STM to separate regions of very smooth growth in the vicinity of the 3C nucleation point from considerably rougher 3C surface regions. These terraces are oriented at right angles to the growth direction. Atomic force microscopy has been used to study etch pits present on the 6H substrate due to high temperature HCl cleaning prior to CVD growth of the 3C layer. The etch pits have hexagonal symmetry and vary in depth from 50 nm to 1 micron.

Potential variations around the grain boundaries (GBs) in antimony (Sb)-doped n-type and boron (B)-doped p-type BaSi₂ epitaxial films on Si(111) were evaluated by Kelvin probe force microscopy. Sb-doped n-BaSi₂ films exhibited positively charged GBs with a downward band bending at the GBs. The average barrier height for holes was approximately 10 meV for an electron concentration n ≈ 10¹⁷ cm⁻³. This downward band bending changed to upward band bending when n was increased to n = 1.8 × 10¹⁸cm⁻³. In the B-doped p-BaSi₂ films, the upward band bending was observed for a hole concentration p ≈ 10¹⁸cm⁻³. The average barrier height for electrons decreased from approximately 25 to 15 meV when p was increased from p = 2.7 × 10¹⁸ to p = 4.0 × 10¹⁸ cm⁻³. These results are explained under the assumption that the position of the Fermi level E{sub f} at GBs depends on the degree of occupancy of defect states at the GBs, while E{sub f} approached the bottom of the conduction band or the top of the valence band in the BaSi₂ grain interiors with increasing impurity concentrations. In both cases, such small barrier heights may not deteriorate the carrier transport properties. The electronic structures of impurity-doped BaSi₂ are also discussed using first-principles pseudopotential method to discuss the insertion sites of impurity atoms and clarify the reason for the observed n-type conduction in the Sb-doped BaSi₂ and p-type conduction in the B-doped BaSi₂.

Hybrid electrode comprising an electric double-layer capacitor of graphene nanosheets and a pseudocapacitor of the electrically conducting polymers namely, polyaniline; PAni and polypyrrole; PPy are constructed that exhibited synergistic effect with excellent electrochemical performance as thin film supercapacitors for alternative energy. The hybrid supercapacitors were prepared by layer-by-layer (LbL) assembly based on controlled electrochemical polymerization followed by reduction of graphene oxide electrochemically producing ErGO, for establishing intimate electronic contact through nanoscale architecture and chemical stability, producing a single bilayer of (PAni/ErGO)1, (PPy/ErGO)1, (PAni/GO)1 and (PPy/GO)1. The rationale design is to create thin films that possess interconnected graphene nanosheets (GNS) with polymer nanostructures forming well-defined tailored interfaces allowing sufficient surface adsorption and faster ion transport due to short diffusion distances. We investigated their electrochemical properties and performance in terms of gravimetric specific capacitance, Cs, from cyclic voltammograms. The LbL-assembled bilayer films exhibited an excellent Cs of ≥350 F g-1 as compared with constituents (˜70 F g-1) at discharge current density of 0.3 A g-1 that outperformed many other hybrid supercapacitors. To gain deeper insights into the physical-chemical interfacial processes occurring at the electrode/electrolyte interface that govern their operation, we have used scanning electrochemical microscopy (SECM) technique in feedback and probe approach modes. We present our findings from viewpoint of reinforcing the role played by heterogeneous electrode surface composed of nanoscale graphene sheets (conducting) and conducting polymers (semiconducting) backbone with ordered polymer chains via higher/lower probe current distribution maps. Also targeted is SECM imaging that allowed to determine electrochemical (re)activity of surface ion adsorption sites

Hybrid electrode comprising an electric double-layer capacitor of graphene nanosheets and a pseudocapacitor of the electrically conducting polymers namely, polyaniline; PAni and polypyrrole; PPy are constructed that exhibited synergistic effect with excellent electrochemical performance as thin film supercapacitors for alternative energy. The hybrid supercapacitors were prepared by layer-by-layer (LbL) assembly based on controlled electrochemical polymerization followed by reduction of graphene oxide electrochemically producing ErGO, for establishing intimate electronic contact through nanoscale architecture and chemical stability, producing a single bilayer of (PAni/ErGO){sub 1}, (PPy/ErGO){sub 1}, (PAni/GO){sub 1} and (PPy/GO){sub 1}. The rationale design is to create thin films that possess interconnected graphene nanosheets (GNS) with polymer nanostructures forming well-defined tailored interfaces allowing sufficient surface adsorption and faster ion transport due to short diffusion distances. We investigated their electrochemical properties and performance in terms of gravimetric specific capacitance, C{sub s}, from cyclic voltammograms. The LbL-assembled bilayer films exhibited an excellent C{sub s} of ≥350 F g{sup −1} as compared with constituents (∼70 F g{sup −1}) at discharge current density of 0.3 A g{sup −1} that outperformed many other hybrid supercapacitors. To gain deeper insights into the physical-chemical interfacial processes occurring at the electrode/electrolyte interface that govern their operation, we have used scanning electrochemical microscopy (SECM) technique in feedback and probe approach modes. We present our findings from viewpoint of reinforcing the role played by heterogeneous electrode surface composed of nanoscale graphene sheets (conducting) and conducting polymers (semiconducting) backbone with ordered polymer chains via higher/lower probe current distribution maps. Also targeted is SECM imaging that allowed to determine

A systematic study of electric transport through thin (2-8 nm) CoFe{sub 2}O{sub 4} films deposited on epitaxial SrRuO{sub 3} bottom electrodes was performed by conducting atomic force microscopy (CAFM). Experimental procedures to investigate transport through thin insulating films by CAFM are critically revised, and the potential of CoFe{sub 2}O{sub 4} films for the use as spin-filtering barriers is assessed. It is concluded that, at room-temperature, a non-tunnel channel significantly contributes to the electric transport, thus limiting the spin-filtering efficiency.

We present here our work on nanoscale wear induced by the tip of an atomic force microscope on thin polystyrene films. Under a wide range of conditions, the repeated scanning of the polymer surface leads to the formation of tip-induced wear patterns consisting of ridges oriented perpendicular to the scanning directions. We found that the evolution of the root mean squared roughness follows an exponential saturation law. Tip-induced wear was more extensive at higher applied loads where transition from rippling to rupturing wear was also observed. Analysis of the patterns formed suggests a crazing mechanism for the observed plastic deformation. The degree of wear as well as the type of the patterns formed was found to depend strongly on the density of the scan lines. In particular an overlap between successive scan lines is necessary to obtain a periodic pattern.

A multispectral imaging method for the rapid and accurate identification of few-layered graphene using optical images is proposed. Commonly rapid identification relies on optical interference effects which limits the choice of substrates and light sources. Our method is based on the comparison of spectral characteristics with principle components from a database which is populated by correlation of micro-Raman registration, spectral characteristics, and optical microscopy. Using this approach the thickness and extent of different graphene layers can be distinguished without the contribution of the optical interference effects and allows characterization of graphene on glass substrates. The high achievable resolution, easy implementation and large scale make this approach suitable for the in-line metrology of industrial graphene production. PMID:25921320

The application of annular bright field (ABF) and medium-angle annular dark field (MAADF) scanning transmission electron microscopy (STEM) imaging to crystalline defect analysis has been extended to dislocations and stacking faults (SFs). Dislocations and SFs have been imaged under zone-axis and two-beam diffraction conditions. Comparing to conventional two-beam diffraction contrast images, the ABF and MAADF images of dislocations and SFs not only are complementary and symmetrical with their peaks at dislocation core and SFs plane, but also show similar extinction phenomenon. It is demonstrated that conventional TEM rules for diffraction contrast, i.e. g · b and g · R invisibility criteria remain applicable. The contrast mechanism and extinction of dislocation and SFs in ABF and MAADF STEM are illuminated by zero-order Laue zone Kikuchi diffraction.

The crystallization of amorphous silicon is studied by transmission electron microscopy. The effect of Ni on the crystallization is studied in a wide temperature range heating thinned samples in-situ inside the microscope. Two cases of limited Ni source and unlimited Ni source are studied and compared. NiSi{sub 2} phase started to form at a temperature as low as 250°C in the limited Ni source case. In-situ observation gives a clear view on the crystallization of silicon through small NiSi{sub 2} grain formation. The same phase is observed at the crystallization front in the unlimited Ni source case, where a second region is also observed with large grains of Ni{sub 3}Si{sub 2}. Low temperature experiments show, that long annealing of amorphous silicon at 410 °C already results in large crystallized Si regions due to the Ni induced crystallization.

Spin polarized low energy electron microscopy has been used to investigate the quantum size effect (QSE) in electron reflectivity from Fe films grown on a pseudomorphic Cu layer on a W(110) surface. Intensity oscillations caused by the QSE as functions of Fe film thickness and incident electron energy identify quantum well resonance conditions in the film. Evaluation of these intensity oscillations using the phase accumulation model provides information on the unoccupied spin polarized band structure in the Fe film above the vacuum level. We also find evidence that the presence of the non-magnetic Cu layer shifts spin polarized quantum well resonances in the Fe layer uniformly downward in energy by 1.1eV compared to Fe/W(110) films without an interface Cu layer, suggesting that the Cu layer gives a small degree of control over the quantum well resonances. PMID:23628648

Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs. PMID:22992750

We review recent studies of double-decker and triple-decker phthalocyanine (Pc) molecules adsorbed on surfaces in terms of the bonding configuration, electronic structure and spin state. The Pc molecule has been studied extensively in surface science. A Pc molecule can contain various metal atoms at the center, and the class of the molecule is called as metal phthalocyanine (MPc). If the center metal has a large radius, like as lanthanoid metals, it becomes difficult to incorporate the metal atom inside of the Pc ring. Pc ligands are placed so as to sandwich the metal atom, where the metal atom is placed out of the Pc plane. The molecule in this configuration is called as a multilayer-decker Pc molecule. After the finding that the double-decker Pc lanthanoid complex shows single-molecule magnet (SMM) behavior, it has attracted a large attention. This is partly due to a rising interest for the ‘molecular spintronics’, in which the freedoms of spin and charge of an electron are applied to the quantum process of information. SMMs represent a class of compounds in which a single molecule behaves as a magnet. The reported blocking temperature, below which a single SMM molecule works as an quantum magnet, has been increasing with the development in the molecular design and synthesis techniques of multiple-decker Pc complex. However, even the bulk properties of these molecules are promising for the use of electronic materials, the films of multi-decker Pc molecules is less studied than those for the MPc molecules. An intriguing structural property is expected for the multi-decker Pc molecules since the Pc planes are linked by metal atoms. This gives an additional degree of freedom to the rotational angle between the two Pc ligands, and they can make a wheel-like symmetric rotation. Due to a simple and well-defined structure of a multi-decker Pc complex, the molecule can be a model molecule for molecular machine studies. The multi-decker Pc molecules can provide

The tissue acetowhitening effect in acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. It is suspected that the optical property (e.g. scattering) change in acetowhitening is due to coagulation of intracellular proteins, but no experimental proof has been reported yet. In this work, we use third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. We studied the acetowhitening effect with different acetic acid concentrations and the co-localized TPEF and THG imaging on tryptophan and NADH at subcellular-level reveals that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope's autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

High-resolution scanning tunneling microscope (STM) images of moire-structured FeO films on Pt(111) were obtained in a number of different tip-dependent imaging modes. For the first time, the STM images are distinguished and interpreted unambiguously with the help of distinct oxygen- vacancy dislocation loops in the FeO moire structure. The experimental STM results are compared with the results of electronic structure calculations within the DFTþUscheme for a realistic (√91 x √ 91)R5.2º moire FeO unit cell supported on Pt(111) as well as with the results from previous studies. We find that one type of STM imaging mode, showing both Fe and O atoms, agrees well with simulated STM images, indicating that the simple Tersoff-Hamann theory is partially valid for this imaging mode. In addition, we identify other distinct, element-specific imaging modes which reveal a strong dependence on the specific tip apex state and likely result from specific tip-sample chemical interactions. From the present STMresults we show that several of the previously published conclusions for the FeO system have to be revisited.

Selected carbon-rich refinery residues ('binders') mixed with mineral particles can form composite materials ('bituminous concrete') with bulk mechanical properties comparable to those of cement concrete. The microstructural mechanism underlying the remarkable composite properties has been related to the appearance of a rigid percolating network consisting of asphaltenes and mineral particles [Wilbrink M. et al. (2005) Rigidity percolation in dispersions with a structured visco-elastic matrix. Phys. Rev. E71, 031402]. In this paper, we explore the microstructure of thin binder films of varying thickness with a number of microscopic characterization techniques, and attempt to relate the observed microstructure to the distinctive mechanical behaviour. Two binders, only one of which has been proven to be suitable for bituminous concrete were investigated, and their microstructure compared. Both binders show the formation of asphaltene aggregates. The binder suitable for bituminous concrete is distinguished by the fact that the asphaltenes show a stronger tendency towards such aggregation, due to a higher concentration and less stabilization in the maltene phase. They also show a clear affinity to other species (such as waxes) and may act as nucleation sites for crystals and aggregates of those species. PMID:21118207

The properties of iron-based superconductors (Fe-SCs) can be varied dramatically with the introduction of dopants and atomic defects. As a pressing example, FeSe, parent phase of the highest-Tc Fe-SC, exhibits prevalent defects with atomic-scale "dumbbell" signatures as imaged by scanning tunneling microscopy (STM). These defects spoil superconductivity when their concentration exceeds 2.5%. Resolving their chemical identity is a prerequisite to applications such as nanoscale patterning of superconducting/nonsuperconducting regions in FeSe as well as fundamental questions such as the mechanism of superconductivity and the path by which the defects destroy it. We use STM and density functional theory to characterize and identify the dumbbell defects. In contrast to previous speculations about Se adsorbates or substitutions, we find that an Fe-site vacancy is the most energetically favorable defect in Se-rich conditions and reproduces our observed STM signature. Our calculations shed light more generally on the nature of Se capping, the removal of Fe vacancies via annealing, and their ordering into a √5 × √5 superstructure in FeSe and related alkali-doped compounds. PMID:27282020

Retention and diffusion of charge in tris(8-hydroxyquinoline) aluminum (Alq(3)) molecular thin films are investigated by injecting electrons and holes via a biased conductive atomic force microscopy tip into the Alq(3) films. After the charge injection, Kelvin force microscopy measurements reveal minimal changes with time in the spatial extent of the trapped charge domains within Alq(3) films, even for high hole and electron densities of >10(12) cm(-2). We show that this finding is consistent with the very low mobility of charge carriers in Alq(3) thin films (<10(-7) cm(2)/(Vs)) and that it can benefit from the use of Alq(3) films as nanosegmented floating gates in flash memory cells. Memory capacitors using Alq(3) molecules as the floating gate are fabricated and measured, showing durability over more than 10(4) program/erase cycles and the hysteresis window of up to 7.8 V, corresponding to stored charge densities as high as 5.4 × 10(13) cm(-2). These results demonstrate the potential for use of molecular films in high storage capacity nonvolatile memory cells. PMID:22332966

The emission of electrons from diamond in vacuum occurs readily as a result of the negative electron affinity of the hydrogenated surface due to features with nanoscale dimensions, which can concentrate electric fields high enough to induce electron emission from them. Electrons can be emitted as a result of an applied electric field (field emission) with possible uses in displays or cold-cathode devices. Alternatively, electrons can be emitted simply by heating the diamond in vacuum to temperatures as low as 350 °C (thermionic emission), and this may find applications in solar energy generation or energy harvesting devices. Electron emission studies usually use doped polycrystalline diamond films deposited onto Si or metallic substrates by chemical vapor deposition, and these films have a rough, faceted morphology on the micron or nanometer scale. Electron emission is often improved by patterning the diamond surface into sharp points or needles, the idea being that the field lines concentrate at the points lowering the barrier for electron emission. However, there is little direct evidence that electrons are emitted from these sharp tips. The few reports in the literature that have studied the emission sites suggested that emission came from the grain boundaries and not the protruding regions. We now present direct observation of the emission sites over a large area of polycrystalline diamond using tunneling atomic force microscopy. We confirm that the emission current comes mostly from the grain boundaries, which is consistent with a model for emission in which the non-diamond phase is the source of electrons with a threshold that is determined by the surrounding hydrogenated diamond surface.

Epitaxial fcc, bcc and hcp metal and alloy films were grown in high vacuum by physical vapour deposition at high rate ('flash' deposition) on the (111), (110) and (100) surfaces of Si and Ge at different deposition temperatures. The resulting epitaxial relationships and morphological features of these films were characterized by transmission electron microscopy and diffraction. Simple epitaxial relationships were found mainly for the fcc metals that form binary eutectic systems with Si and G e. Of these, Ag exhibited exceptional behaviour by forming in a single crystal cube-cube relationship on all six semiconductor surfaces. Al and Au both formed bicrystal films on (100) substrates but differed in their behaviours on (111) substrates. Silicide formers such as the fcc metals Cu and Ni, as well as all bcc and hcp metals investigated, did not adopt epitaxial relationships on most semiconductor substrates. However, epitaxial single-crystal, bicrystal and tricrystal films of several metals and alloys could be grown by using a Ag buffer layer. The factors controlling the epitaxial growth of metal films are discussed in the light of the observations and compared with the predictions of established models for epitaxial relationships. It is concluded that epitaxial films can be grown easily if the film forms a simple eutectic or monotectic system with the substrate. The epitaxial relationships of those films depend on crystallographic factors for metal-metal epitaxy and on the substrate surface structure for metal-semiconductor epitaxy.

We investigated how stress relaxation mapping is quantified compared with the force modulation mapping of confluent epithelial cells using atomic force microscopy (AFM). Using a multi-frequency AFM technique, we estimated the power-law rheological behaviors of cells simultaneously in time and frequency domains. When the power-law exponent α was low (<0.1), the α values were almost the same in time and frequency domains. On the other hand, we found that at the high values (α > 0.1), α in the time domain was underestimated relative to that in the frequency domain, and the difference increased with α, whereas the cell modulus was overestimated in the time domain. These results indicate that power-law rheological parameters estimated by stress relaxation are sensitive to lag time during initial indentation, which is inevitable in time-domain AFM experiments.

Identification of proliferating chondrocytes along the periphery of laser ablation sites in irradiated cartilage has led to interest in studying the use of laser heating alone to stimulate chondrocyte growth. However, excessive heat produced by a laser can also cause chondrocyte necrosis and apoptosis. The objective of this study was to evaluate acute injury to cartilage following irradiation by an Nd:YAG (λ=1.32μm) laser in intact ex-vivo tissue specimens. Rabbit nasal septal cartilage was irradiated using an Nd:YAG laser using pulse durations (4, 6, and 8 seconds) and power (4, 6, and 8 watts) settings previously determined to produce cell division. Immediately after laser irradiation, the extent of thermal injury to the cartilage samples was evaluated using a Live/Dead cell viability assay combined with confocal microscopy. Thermal injury was assessed with respect to distribution of live and dead cells surrounding the laser spot where regeneration was previously observed. The cell viability assay identified necrotic tissue within and immediately around the laser spot. Moving away from the center of the laser spot, a mixed population of necrotic and live chondrocytes was observed. As expected, a correlation between irradiation time, power and degree of injury was found. The results of this experiment will be used to determine the threshold required to produce regeneration while minimizing thermal injury.

We study the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degrees of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/noninvasive), and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy is used for high-resolution topography and stiffness imaging of actin filaments within living cells. In healthy cells, local stiffness is maximum where filamentous actin is organized as well-aligned stress fibers, resulting in apparent Young's modulus values up to 1 order of magnitude larger than those in regions where these structures are not observed, but these organized actin fibers are barely observed in tumorigenic cells. We further investigate cytoskeleton conformation in the three cell lines by immunofluorescence confocal microscopy. The combination of both techniques determines that actin stress fibers are present at apical regions of healthy cells, while in tumorigenic cells they appear only at basal regions, where they cannot contribute to stiffness as probed by atomic force microscopy. These results substantiate that actin stress fibers provide a dominant contribution to stiffness in healthy cells, while the elasticity of tumorigenic cells appears not predominantly determined by these structures. We also discuss the effects of the high-frequency indentations inherent to peak-force atomic force microscopy for the identification of mechanical cancer biomarkers. Whereas conventional low loading rate indentations (1 Hz) result in slightly differentiated average stiffness for each cell line, in high-frequency measurements (250 Hz) healthy cells are clearly discernible from both tumorigenic cells with an enhanced stiffness ratio; however, the two cancerous cell lines produced indistinguishable results. PMID:26901115

Microstructure and electronic structure of highly strained bismuth ferrite (BiFeO3) thin films grown on lanthanum aluminate substrates are studied using high-resolution transmission and scanning transmission electron microscopies and electron energy loss spectroscopy (EELS). Monoclinic and tetragonal phases were observed in films grown at different temperatures, and a mix of both phases was detected in a film grown at intermediate temperature. In this film, a smooth transition of the microstructure was found between the monoclinic and the tetragonal phases. A considerable increase in the c-axis parameters was observed in both phases compared with the rhombohedral bulk phase. The off-center displacement of iron (Fe) ions was increased in the monoclinic phase as compared with the tetragonal phase. EEL spectra show different electronic structures in the monoclinic and the tetragonal phases. These experimental observations are well consistent with the results of theoretical first-principle calculations performed.

Microstructure and electronic structure of highly strained bismuth ferrite (BiFeO{sub 3}) thin films grown on lanthanum aluminate substrates are studied using high-resolution transmission and scanning transmission electron microscopies and electron energy loss spectroscopy (EELS). Monoclinic and tetragonal phases were observed in films grown at different temperatures, and a mix of both phases was detected in a film grown at intermediate temperature. In this film, a smooth transition of the microstructure was found between the monoclinic and the tetragonal phases. A considerable increase in the c-axis parameters was observed in both phases compared with the rhombohedral bulk phase. The off-center displacement of iron (Fe) ions was increased in the monoclinic phase as compared with the tetragonal phase. EEL spectra show different electronic structures in the monoclinic and the tetragonal phases. These experimental observations are well consistent with the results of theoretical first-principle calculations performed.

The photothermal optical lock-in optical coherence microscope (poli-OCM) introduced molecular specificity to OCM imaging, which is conventionally, a label-free technique. Here we achieve three-dimensional live cell and mitochondria specific imaging using ~4nm protein-functionalized gold nanoparticles (AuNPs). These nanoparticles do not photobleach and we demonstrate they're suitability for long-term time lapse imaging. We compare the accuracy of labelling with these AuNPs using classical fluorescence confocal imaging with a standard mitochondria specific marker. Furthermore, time lapse poli-OCM imaging every 5 minutes over 1.5 hours period was achieved, revealing the ability for three-dimensional monitoring of mitochondria dynamics.

Dendritic cells are professional phagocytes that are highly specialized to process and present antigens from internalized particles to prime naïve T cells. To achieve their functions, the phagocytic machinery and membrane dynamics of these cells have been adapted to optimize presentation of antigens from phagocytosed particles that bear ligands of pattern recognition receptors, such as toll-like receptors (TLRs), and that are thus perceived of as “dangerous”. We have recently shown that phagosomes that are engaged in TLR signaling in dendritic cells emit numerous long tubules that facilitate content exchange with other signaling phagosomes and favor presentation of particle-derived antigens. This chapter describes the methods used to study the formation of these tubules, which we refer to as “phagotubules”, by live cell imaging of mouse dendritic cells after the phagocytosis of fluorescent latex beads. We also describe methods to assess the effect of TLR signaling on this process. PMID:25702119

We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the quantitative study of cellular dynamics. Living cells in culture are optically probed by measuring the phase shift they produce on the transmitted wave front. The high temporal stability of the phase signal, equivalent to λ/1800, and the low acquisition time (~20μs) enable to monitor cellular dynamics processes. An experimental procedure allowing to calculate both the integral refractive index and the cellular thickness (morphometry) from the measured phase shift is presented. Specifically, the method has been applied to study the dynamics of neurons in culture during a hypotonic stress. Such stress produces a paradoxical decrease of the phase which can be entirely resolved by applying the methodological approach described in this article; indeed the method allows to determine independently the thickness and the integral refractive index of cells.

Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines. PMID:27576709

Mucus transport is essential to remove inhaled particles and pathogens from the lung. Impaired removal of mucus often results in worsening of lung diseases. To understand the mechanisms of mucus transport and to monitor the impact of therapeutic strategies, it is essential to visualize airways and mucus in living animals without disturbing transport processes by intubation or surgically opening the airways. We developed a custom-built optical coherence microscope (OCM) providing a lateral and axial resolution of approximately 1.5 µm with a field of view of 2 mm at up to 150 images/s. Images of the intact trachea and its mucus transport were recorded in anesthetized spontaneously breathing mice. NaCl solution (0.9% and 7%) or Lipopolysaccharide were applied intranasally. OCM resolved detailed structure of the trachea and enabled measuring the airway surface liquid (ASL) thickness through the tracheal wall. Without stimulation, the amount of ASL was only a few µm above the epithelium and remained constant. After intranasal application of 30 µl saline at different concentrations, an early fast cough-like fluid removal with velocities higher than 1 mm/s was observed that removed a high amount of liquid. The ASL thickness increased transiently and quickly returned to levels before stimulation. In contrast to saline, application of Lipopolysaccharide induced substantial mucus release and an additional slow mucus transport by ciliary beating (around 100 µm/s) towards the larynx was observed. In conclusion, OCM is appropriate unique tool to study mechanisms of mucus transport in the airways and effects of therapeutic interventions in living animals.

In this work, a method based on scanning Kelvin probe microscopy is proposed to separately extract source/drain (S/D) series resistance in operating amorphous indium–gallium–zinc–oxide (a-IGZO) thin-film transistors. The asymmetry behavior of S/D contact resistance is deduced and the underlying physics is discussed. The present results suggest that the asymmetry of S/D contact resistance is caused by the difference in bias conditions of the Schottky-like junction at the contact interface induced by the parasitic reaction between contact metal and a-IGZO. The overall contact resistance should be determined by both the bulk channel resistance of the contact region and the interface properties of the metal-semiconductor junction. Project supported by the Key Industrial R&D Program of Jiangsu Province, China (Grant No. BE2015155), the Priority Academic Program Development of Higher Education Institutions of Jiangsu Province, China, and the Fundamental Research Funds for the Central Universities, China (Grant No. 021014380033).

For rhombohedral multiferroelectrics, non-180° ferroelectric domain switching may induce ferroelastic and/or (anti-)ferromagnetic effect. So the determination and control of ferroelectric domain switching angles is crucial for nonvolatile information storage and exchange-coupled magnetoelectric devices. We try to study the intrinsic characters of polarization switching in BiFeO3 by introducing a special data processing method to determine the switching angle from 2D PFM (Piezoresponse Force Microscopy) images of randomly oriented samples. The response surface of BiFeO3 is first plotted using the piezoelectric tensor got from first principles calculations. Then from the normalized 2D PFM signals before and after switching, the switching angles of randomly oriented BiFeO3 grains can be determined through numerical calculations. In the polycrystalline BiFeO3 films, up to 34% of all switched area is that with original out-of-plane (OP) polarization parallel to the poling field. 71° polarization switching is more favorable, with the area percentages of 71°, 109° and 180° domain switching being about 42%, 29% and 29%, respectively. Our analysis further reveals that IP stress and charge migration have comparable effect on switching, and they are sensitive to the geometric arrangements. This work helps exploring a route to control polarization switching in BiFeO3, so as to realize desirable magnetoelectric coupling. PMID:26192555

For rhombohedral multiferroelectrics, non-180° ferroelectric domain switching may induce ferroelastic and/or (anti-)ferromagnetic effect. So the determination and control of ferroelectric domain switching angles is crucial for nonvolatile information storage and exchange-coupled magnetoelectric devices. We try to study the intrinsic characters of polarization switching in BiFeO3 by introducing a special data processing method to determine the switching angle from 2D PFM (Piezoresponse Force Microscopy) images of randomly oriented samples. The response surface of BiFeO3 is first plotted using the piezoelectric tensor got from first principles calculations. Then from the normalized 2D PFM signals before and after switching, the switching angles of randomly oriented BiFeO3 grains can be determined through numerical calculations. In the polycrystalline BiFeO3 films, up to 34% of all switched area is that with original out-of-plane (OP) polarization parallel to the poling field. 71° polarization switching is more favorable, with the area percentages of 71°, 109° and 180° domain switching being about 42%, 29% and 29%, respectively. Our analysis further reveals that IP stress and charge migration have comparable effect on switching, and they are sensitive to the geometric arrangements. This work helps exploring a route to control polarization switching in BiFeO3, so as to realize desirable magnetoelectric coupling.

A nanoscale linked-crater structure was fabricated on an Al surface by chemical and electrochemical combination processes. The surface of an Al plate was treated with Semi Clean and was successively processed in anodization in H IISO 4. Dynamic force microscopy image (DFM) showed that a linked-crater structure was formed on the Al surface. At the next stage, the authors conducted the thin film growth of conducting polymer polythiophene on the Al surface by an electrochemical method. The electrochemical polymerization on the Al surface was performed in acetonitrile containing thiophene monomer and (Et) 4NBF 4 as a supporting electrolyte. After being electrochemically processed, the contour image of each crater was still recognized implying that the polymer nanofilm was grown on the nanoscale structured Al surface. The cross section analysis demonstrated that the nanofilm was grown along the linked-crater structure because the contour of each crater became thick. X-ray photoemission spectroscopy measurement also supported the polymer nanofilm growth because C 1s and S 2p lines were detected. Furthermore, copper phthalocyanine (CuPc) molecules are injected into the polymer nanofilm grown on the nanoscale structured Al surface by diffusing method in order to functionalize the nanoscale hybrid material.

For rhombohedral multiferroelectrics, non-180° ferroelectric domain switching may induce ferroelastic and/or (anti-)ferromagnetic effect. So the determination and control of ferroelectric domain switching angles is crucial for nonvolatile information storage and exchange-coupled magnetoelectric devices. We try to study the intrinsic characters of polarization switching in BiFeO3 by introducing a special data processing method to determine the switching angle from 2D PFM (Piezoresponse Force Microscopy) images of randomly oriented samples. The response surface of BiFeO3 is first plotted using the piezoelectric tensor got from first principles calculations. Then from the normalized 2D PFM signals before and after switching, the switching angles of randomly oriented BiFeO3 grains can be determined through numerical calculations. In the polycrystalline BiFeO3 films, up to 34% of all switched area is that with original out-of-plane (OP) polarization parallel to the poling field. 71° polarization switching is more favorable, with the area percentages of 71°, 109° and 180° domain switching being about 42%, 29% and 29%, respectively. Our analysis further reveals that IP stress and charge migration have comparable effect on switching, and they are sensitive to the geometric arrangements. This work helps exploring a route to control polarization switching in BiFeO3, so as to realize desirable magnetoelectric coupling. PMID:26192555

The resistance R versus perpendicular external magnetic field H was measured for superconducting Nb thin film microbridges with and without microholes (antidots, ADs). Well below the transition temperature, integral R(H) measurements of the resistive transition to the normal state on the plain bridge show two distinct regions, which can be identified as bulk and edge superconductivity, respectively. The latter case appears when bulk superconductivity becomes suppressed at the upper critical field Hc2 and below the critical field of edge superconductivity Hc3 ≈ 1.7 Hc2. The presence of additional edges in the AD bridge leads to a different shape of the R(H) curves. We used low-temperature scanning laser microscopy (LTSLM) to visualize the current distribution in the plain and AD bridges upon sweeping H. While the plain bridge shows a dominant LTSLM signal at its edges for H > Hc2 the AD bridge also gives a signal from the inner parts of the bridge due to the additional edge states around the ADs. LTSLM reveals an asymmetry in the current distribution between the left and right edges, which confirms theoretical predictions. Furthermore, the experimental results are in good agreement with our numerical simulations (based on the time-dependent Ginzburg-Landau model), yielding the spatial distribution of the order parameter and current density for different bias currents and H values.

Conventional evaluation methods of chemotherapeutic efficacy such as tissue biopsy and anatomical measurement are either invasive with potential complications or dilatory to capture the rapid pathological changes. Here, a sensitive and resolution-scalable photoacoustic microscopy (PAM) with theranostic nanoformulation was developed to noninvasively monitor the therapy response in a timely manner. Ultrasmall graphene oxide nanosheets were designed as both drug-loading vehicle and photoacoustic signal amplifier to the tumor. With the signal enhancement by the injected contrast agents, the subtle microvascular changes of the chemotherapy response in tumor were advantagely revealed by our PAM system, which was much earlier than the morphological measurement by standard imaging techniques. High tumor uptake of the enhanced nanodrug with Cy5.5 labeling was validated by fluorescence imaging. At different observation scales, PAM offered unprecedented sensitivity of optical absorption and high spatial resolution over optical imaging. Our studies demonstrate the PAM system with synergistic theranostic strategy to be a multiplexing platform for tumor diagnosis, drug delivery, and chemotherapy response monitoring at a very early stage and in an effective way. PMID:25406986

We have investigated the thickness and surface structure of surface freezing films in Ga-Bi and Ga-Pb alloys over a wide temperature range between room temperature and the respective surface freezing transitions by x-ray photoelectron spectroscopy (XPS) and scanning tunnelling microscopy (STM). For the example of a Ga-Bi alloy dilute in Bi, XPS measurements show that the surface freezing film has a nearly constant value of approximately 25 A between the surface freezing temperature of 130 degrees C and room temperature if the sample is cooled slowly (5 Kh). On heating to 130 degrees C the film thickness exhibits a clear hysteresis on melting. On quenching the alloy sample (>100 Kh) the film thickness increases by almost a factor of 10. These observations indicate that the surface freezing films are metastable. The surface structure of the surface freezing films of various Ga-rich Ga-Bi and Ga-Pb alloys has been probed for the first time by STM at different temperatures below and above the bulk eutectic point. Atomically resolved STM images show the surface structures of pure Bi (0001) and Pb (111), respectively, at room temperature. On heating above the eutectic temperature the surface structure of the films does not change significantly as judged from the size and thickness of Pb or Bi terraces. These observations together with the film thickness variation with temperature indicate that the surface freezing films behave like a metastable independent surface phase. These results together with the wetting characteristics of these alloys suggest that surface freezing in these systems is a first order surface phase transition between wetting and metastable surface freezing films. The energy barrier for nucleation is strongly reduced due to a lowering of the interfacial energy if the nucleus is completely immersed in the respective wetting layer. PMID:17655450

A plurality of articles discussing combined effects of acoustic high-pressure (mechanical factor) and heat (thermal factor) caused by acoustic vibration on biological tissues and cells has been published. Herein, we contribute the preliminary results describing the behavior of living human skin cells when separately applying shock waves and thermal insult to them. First, we gradually increased temperature of a culturing medium from 37.5 to 52 degree(s)C using the heat plate with temperature controller, and carried out in-situ observation of the cells grown on a substrate via the medium using a scanning acoustic microscope. Second, we provided the pressure using high power ultrasonic pulses generated by a laser induced ultrasonic shock wave system to the cells, wherein the pressure caused by the pulses was measured by a hydrophone, and wherein temperature was monitored by thermocouples. The cells were observed just after giving the impact. The difference between phenomena indicating cellular insult and injury (e.g., shrinkage or lift-off) were clearly visualized by the scanning acoustic microscope with frequency at 1.0 GHz.

Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system. PMID:12885672

We investigated the surface potential of the ferroelectric domains of the epitaxial PbTiO{sub 3} (PTO) films using both Kelvin probe and piezoresponse force microscopy. The surface potential changes as a function of applied biases suggested that the amount and sign of surface potentials depend on the correlation between polarization and screen charges. It also suggested that the trapped negative charges exist on the as-deposited PTO surfaces. Injected charges and their resultant surface potentials are investigated by grounded tip scans. The results unveiled the origin of surface potential changes during ferroelectric switching in the epitaxial PTO films.

An innovative route for deep-submicrometer spatial resolution hard x-ray microscopy with tabletop x-ray source is proposed. A film of lithium fluoride (LiF) was used as imaging detector in contact mode. We present here the x-ray images recorded on LiF films of a Fresnel zone plate with submicrometer gold structures and of an onion cataphyll. The images were read with an optical confocal microscope in fluorescence mode. The measured spatial resolution was about 250 nm, i.e., close to the resolution limit of the confocal microscope. The advantages and drawbacks, and the possible improvements, of this route are discussed.

An innovative route for deep-submicrometer spatial resolution hard x-ray microscopy with tabletop x-ray source is proposed. A film of lithium fluoride (LiF) was used as imaging detector in contact mode. We present here the x-ray images recorded on LiF films of a Fresnel zone plate with submicrometer gold structures and of an onion cataphyll. The images were read with an optical confocal microscope in fluorescence mode. The measured spatial resolution was about 250nm, i.e., close to the resolution limit of the confocal microscope. The advantages and drawbacks, and the possible improvements, of this route are discussed.

Coherent anti-Stokes Raman scattering (CARS) microscopy can be used as a powerful imaging technique to identify chemical compositions of complex samples in biology, biophysics, medicine, and materials science. In this work we developed a CARS microscopic system capable of hyperspectral imaging. By employing an ultrafast laser source, a photonic crystal fiber, and a scanning laser microscope together with spectral detection by a highly sensitive back-illuminated cooled charge-coupled device (CCD) camera, we were able to rapidly acquire and process hyperspectral images of live cells with chemical selectivity. We discuss various aspects of hyperspectral CARS image analysis and demonstrate the use of singular value decomposition methods to characterize the cellular lipid content. PMID:25198903

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos–c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development. PMID:26303532

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development. PMID:26303532

The degree of mechanical stiffness of vascular endothelial cells determines the endogenous production of the vasodilating gas nitric oxide (NO). However, the underlying mechanisms are not yet understood. Experiments on vascular endothelial cells suggest that the electrical plasma membrane potential is involved in this regulatory process. To test this hypothesis we developed a technique that simultaneously measures the electrical membrane potential and stiffness of vascular endothelial cells (GM7373 cell line derived from bovine aortic endothelium) under continuous perfusion with physiological electrolyte solution. The cellular stiffness was determined by nano-indentation using an atomic force microscope (AFM) while the electrical membrane potential was measured with bis-oxonol, a voltage-reporting fluorescent dye. These two methods were combined using an AFM attached to an epifluorescence microscope. The electrical membrane potential and mechanical stiffness of the same cell were continuously recorded for a time span of 5 min. Fast fluctuations (in the range of seconds) of both the electrical membrane potential and mechanical stiffness could be observed that were not related to each other. In contrast, slow cell depolarizations (in the range of minutes) were paralleled by significant increases in mechanical stiffness. In conclusion, using the combined AFM-fluorescence technique we monitored for the first time simultaneously the electrical plasma membrane potential and mechanical stiffness in a living cell. Vascular endothelial cells exhibit oscillatory non-synchronized waves of electrical potential and mechanical stiffness. The sustained membrane depolarization, however, is paralleled by a concomitant increase of cell stiffness. The described method is applicable for any fluorophore, which opens new perspectives in biomedical research.

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content. PMID:22947854

Total-internal-reflection fluorescence (TIRF) microscopy provides high optical-sectioning capability and a good signal-contrast ratio for structures near the surfaces of cells. In recent years, several improvements have been developed, such as variable-angle TIRF (VA-TIRF) and spinning TIRF (sp-TIRF), which permit quantitative image analysis and address non-uniform scattering fringes, respectively. Here, we present a dual-color DMD-based shadowless-illuminated variable-angle TIRF (siva-TIRF) system that provides a uniform illumination field. By adjusting the incidence angle of the illuminating laser on the back focal plane (BFP) of the objective, we can rapidly illuminate biological samples in layers of various thicknesses in TIRF or hollow-cone epi-fluorescence mode. Compared with other methods of accomplishing VA-TIRF/sp-TIRF illumination, our system is simple to build and cost-effective, and it provides optimal multi-plane dual-color images. By showing spatiotemporal correlated movement of clathrin-coated structures with microtubule filaments from various layers of live cells, we demonstrate that cortical microtubules are important spatial regulators of clathrin-coated structures. Moreover, our system can be used to prove superb axial information of three-dimensional movement of structures near the plasma membrane within live cells. PMID:24877013

Quantum dots (Qdots) have become ubiquitous in biomedical research due to their excellent brightness, photostability, monodispersity, and fluorescent yield. Furthermore, they have become increasingly useful as imaging agents which are valuable for answering molecular questions in living subjects. However, little is currently known about how nanoparticles such as Qdots interact at the microscale within the vasculature and tumor microenvironments in living subjects. In order to further our understanding of the dynamic processes involved in Qdot targeting in the intact tumor, we developed an in vivo binding assay to visualize and fully elucidate this approach using a variety of animal models and tumor types. We employed argine-glycine-aspartic acid (RGD) peptides to specifically target the αvβ3 integrins which are expressed on the surface of endothelial cells comprising newly formed or forming blood vessels; RGD peptides were conjugated to the Qdot surface. Exploiting intravital microscopy with subcellular-level resolution, we directly observed and recorded the binding of nanoparticle conjugates in two different murine models, using three different tumor cell lines. Using this generalizable approach, we learned that RGD-qdots unexpectedly bind to tumor blood vessels in all models tested only as aggregates rather than individually. Understanding these issues on the microscale using such techniques will provide a platform for the rational design of molecularly-targeted nanoparticles including Qdots. This is critical for nanoparticles to become a valuable research tool with the potential to become clinically valuable imaging and therapeutic agents, particularly for ensuring regulatory approval of such nanoparticles.

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

Picosecond fluorescence lifetime imaging microscopy (FLIM) provides a most valuable tool to analyze the primary processes of photosynthesis in individual cells and chloroplasts of living cells. In order to obtain correct lifetimes of the excited states, the peak intensity of the exciting laser pulses as well as the average intensity has to be sufficiently low to avoid distortions of the kinetics by processes such as singlet-singlet annihilation, closing of the reaction centers or photoinhibition. In the present study this requirement is achieved by non-scanning wide-field FLIM based on time- and space-correlated single-photon counting (TSCSPC) using a novel microchannel plate photomultiplier with quadrant anode (QA-MCP) that allows parallel acquisition of time-resolved images under minimally invasive low-excitation conditions. The potential of the wide-field TCSPC method is demonstrated by presenting results obtained from measurements of the fluorescence dynamics in individual chloroplasts of moss leaves and living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Estradiol is a steroid hormone that binds and activates estradiol receptors. Activation of these receptors is known to modulate neuronal physiology and provide neuroprotection, but it is not completely understood how estradiol mediates these actions on the nervous system. Activation of a sub-population of estradiol receptor-α (ERα), originally identified as a nuclear protein, localizes to the plasma membrane and appears to be a critical step in neuroprotection against brain injury and disease. Previously we showed that estradiol stimulates the rapid and transient trafficking of plasma membrane ERα in primary hypothalamic neurons, and internalization of membrane-impermeant estradiol (E6BSA-FITC) into cortical neuron endosomes in vitro. These findings support the concept that estradiol activates and down-regulates plasma membrane ERα by triggering endocytosis. Here, we use TIRFM (total internal reflection fluorescence microscopy) to image the trafficking of E6BSA-FITC, and GFP-labeled ERα, in live cells in real time. We show that activation of plasma membrane ERs by E6BSA-FITC result in internalization of the fluorescent ligand in live N-38 neurons, an immortalized hypothalamic cell line. Pretreatment with ER antagonist ICI 182,780 decreased the number of E6BSA-FITC labeled puncta observed. We also observed in live N-38 neurons that E6BSA-FITC co-localized with FM4-64 and LysoTracker fluorescent dyes that label endosomes and lysosomes. Our results provide further evidence that plasma membrane ERα activation results in endocytosis of the receptor. PMID:24353903

Changes in the morphology and conductance state of [(GeTe)2(Sb2Te3)] superlattice (SL) films on Si(100) caused by external voltage were investigated by multimode scanning probe microscopy (MSPM) and scanning probe lithography (SPL) at room temperature in vacuum. After SPL patterning at a write voltage exceeding a threshold value, grain-dependent changes in transverse film conductance appeared in the MSPM current maps at a low voltage. Specific details of the conductance state switching were dependent on the film growth process. In uniform films grown in a two-step process, a threshold voltage of 1.6 V and a minimum switching power of ˜15 pW were obtained for conductance switching activated by high-energy electrons injected from the probe. Above 3.0 V, thermally driven regrowth of the SL films was observed. The results demonstrate a simple and appropriate method of optimizing topological SL films as recording media without device fabrication.

The growth of water on thin SiO{sub 2} films on Si wafers at vapor pressures between 1.5 and 4 torr and temperatures between -10 and 21 C has been studied in situ using Kelvin Probe Microscopy and X-ray photoemission and absorption spectroscopies. From 0 to 75% relative humidity (RH) water adsorbs forming a uniform film 4-5 layers thick. The surface potential increases in that RH range by about 400 mV and remains constant upon further increase of the RH. Above 75% RH the water film grows rapidly, reaching 6-7 monolayers at around 90% RH and forming a macroscopic drop near 100%. The O K-edge near-edge X-ray absorption spectrum around 75% RH is similar to that of liquid water (imperfect H-bonding coordination) at temperatures above 0 C and ice-like below 0 C.

The initial oxidation behavior of Cu 0.5Au 0.5 (0 0 1) thin film was investigated by in situ ultrahigh vacuum transmission electron microscopy to model nano-oxidation of alloys with one active component and one noble component. The formation of irregular-shaped octahedron Cu 2O islands with cube-on-cube crystallographic orientation to the substrate film was observed at all temperature studied. The energetics of Cu 2O nucleation for Cu and Cu 0.5Au 0.5 oxidation was compared. Cu 0.5Au 0.5 oxidation has lower nucleation activation energy due to the reduced mismatch strain between Cu 2O and Cu 0.5Au 0.5 films. On the other hand, the reaction kinetics for Cu 0.5Au 0.5 alloy oxidation is slower due to the higher diffusion activation energy of Cu.

Cross-sections of laser fabricated nanosharp tips and microbumps on silicon and metal thin films are produced and examined in this work. These structures are formed with a Q-switched neodymium doped yttrium aluminum garnet nanosecond-pulse laser, emitting at its fourth harmonic of 266 nm, using a mask projection technique to generate circular laser spots, several microns in diameter. Cross-section of selected structures were produced using a focused ion beam and were characterized via electron microscopy. The diffraction patterns of the silicon samples indicate that the laser formed tip maintains the same single crystal structure as the original silicon film. Examinations of the laser formed structures in metal films confirm that the microbumps are hollow, while revealing that the vertical protrusions are solid.

FeMn/Fe/Co/Cu(1,1,10) films were grown epitaxially and investigated using the magneto-optic Kerr effect and photoemission electron microscopy. We found that FeMn/Fe/Co/Cu(1,1,10) exhibits the same properties as FeMn/Co/Cu(1,1,10) for the ferromagnetic phase of the face centered cubic (fcc) Fe film but a different property for the non-ferromagnetic phase of the fcc Fe film. This result indicates that the characteristic property reported in the literature for FeMn/Co/Cu(001) comes from the FeMn spin structure and is independent of the ferromagnetic layer.

TiO{sub 2} thin films grown on (11{bar 2}0) sapphire at 800{degree}C by the MOCVD technique have been characterized by transmission electron microscopy. The TiO{sub 2} thin films are single crystalline and have the rutile structure. The epitaxial orientation relationship between the TiO{sub 2} thin films (R) and the substrate (S) has been found to be: (101)(0{bar 1}0){sub R}{parallel}(11{bar 2}0)(0001){sub S}. Growth twins in the films are commonly observed with the twin plane {l brace}101{r brace} and twinning direction {l angle}011{r angle}. Detailed atomic structures of the twin boundaries and TiO{sub 2}/{alpha}-Al{sub 2}O{sub 3} interfaces have been investigated by high-resolution electron microscopy (HREM). When the interfaces are viewed in the direction of (0{bar 1}0){sub R}/(0001){sub S}, the interfaces are found to be structurally coherent in the direction of ({bar 1}01){sub R}/(1{bar 1}00){sub S}, in which the lattice mismatch at the interfaces is about 0.5%. 8 refs., 4 figs.

We developed a technique that uses scanning probe microscopy (SPM) to study the real-time injection and extraction of charge carriers in thin film devices. We investigate the effects of oxidation on thin films of Lead Sulfide (PbS) quantum dots with tetrabutyl-ammonium-iodide (TBAI) ligands in an inverted field effect transistor geometry with gold electrodes. By positioning the SPM tip at an individual location and using Kelvin Probe Force Microscopy (KPFM) to measure the potential over time, we can record how the charge carriers respond to changing the backgate voltage with grounded source and drain electrodes. We see relatively fast screening for negative backgate voltages because holes are quickly injected into the PbS film. The screening is slower for positive gate voltages, because some of these holes are trapped and therefore less mobile. We probe these trapped holes by applying different gate voltages and recording the change in potential at the surface. There are mixed reports about the effect of air exposure on thin films of PbS quantum dots, with initial exposure appearing to be beneficial to device characteristics. We study the change in current, mobility, and charge injection and extraction as measured by KPFM over hours and days of exposure to air. This work is supported by NSF Grant DMR-0955348, and the Center for Heirarchical Manufacturing at the University of Massachusetts, Amherst (NSF CMMI-1025020).

Pd/SiC has been used as a hydrogen and a hydrocarbon gas sensor operated at high temperature. UHV (Ultra High Vacuum)-Scanning Tunneling Microscopy (STM), Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS) techniques were applied to study the relationship between the morphology and chemical compositions for Pd ultra-thin films on SiC (less than 30 angstroms) at different annealing temperatures. Pd ultra-thin film on 6H-SiC was prepared by the RF sputtering method. The morphology from UHV-STM and AFM shows that the Pd thin film was well deposited on SiC substrate, and the Pd was partially aggregated to round shaped participates at an annealing temperature of 300 C. At 400 C, the amount of surface participates decreases, and some strap shape participates appear. From XPS, Pd2Si was formed on the surface after annealing at 300 C, and all Pd reacted with SiC to form Pd2Si after annealing at 400 C. The intensity of the XPS Pd peak decreases enormously at 400 C. The Pd film diffused into SiC, and the Schottky barrier height has almost no changes. The work shows the Pd sicilides/SiC have the same electronic properties with Pd/SiC, and explains why the Pd/SiC sensor still responds to hydrogen at high operating temperatures.

In this article, the authors describe the development and dissemination of a film-based educational intervention to reduce negative attitudes toward gay and bisexual men, transgender women, and people living with HIV/AIDS in Latino communities, with a focus on youth. The intervention, Tal Como Somos/Just as We Are, is based on stigma and attribution theories, extensive formative research, and community input. Evaluation findings among educators and school youth suggest the film has the potential to effectively influence attitudes toward gay and bisexual men, transgender women, and people living with HIV/AIDS. The film and intervention are being disseminated using diffusion of innovations theory through community-based organizations, schools, television broadcasting, and film festivals. PMID:24377496

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM. PMID:25786736

In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica JCM10317 (PaE) was analyzed by surface plasmon resonance (SPR). The adsorption of PaE and the degradation rate for polyester films were quantitatively monitored by a positive and negative SPR signal shifts, respectively. The decrease in SPR signal and the erosion depth of amorphous poly(L-lactide) (a-PLLA) film measured by atomic force microscopy (AFM) had a linear relationship, and the weight loss was estimated from the AFM data combined with a density of a-PLLA film. Furthermore, SPR sensorgrams for various polyester films showed that degradation rate of poly(ε-caprolactone) and poly(butylene succinate-co-adipate) which contain C6 units was higher than that of other polyesters such as poly(butylene succinate) and a-PLLA. These results suggest that C6 is the preferred chain length as substrates for PaE. PMID:23339012

A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-resolution images of specific proteins and their complexes. Auto-fluorescence however, can severely compromise image contrast and represents a fundamental limitation for imaging proteins within living cells. We have previously shown that optical switch probes and optical lock-in detection (OLID) image microscopy improve image contrast in high background environments. Here, we present the design, synthesis and characterization of amino- reactive and cell permeable optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR-NISO undergoes rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with 365 nm and >530 nm light. In the MC-state, the TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET) to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal. Irradiation of TMR-NISO with a defined sequence of 365 nm and 546 nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High fidelity optical switching of TMR fluorescence is shown for TMR-NISO probes in vitro and for membrane permeable TMR-NISO within living cells. PMID:20674372

In-vivo microscopic long term time-lapse studies require controlled imaging conditions to preserve sample viability. Therefore it is crucial to meet specific exposure conditions as these may limit the applicability of established techniques. In this work we demonstrate the use of third harmonic generation (THG) microscopy for long term time-lapse three-dimensional studies (4D) in living Caenorhabditis elegans embryos employing a 1550 nm femtosecond fiber laser. We take advantage of the fact that THG only requires the existence of interfaces to generate signal or a change in the refractive index or in the χ3 nonlinear coefficient, therefore no markers are required. In addition, by using this wavelength the emitted THG signal is generated at visible wavelengths (516 nm) enabling the use of standard collection optics and detectors operating near their maximum efficiency. This enables the reduction of the incident light intensity at the sample plane allowing to image the sample for several hours. THG signal is obtained through all embryo development stages, providing different tissue/structure information. By means of control samples, we demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Certainly, this technique reduces the complexity of sample preparation (i.e. genetic modification) required by established linear and nonlinear fluorescence based techniques. We demonstrate the non-invasiveness, reduced specimen interference, and strong potential of this particular wavelength to be used to perform long-term 4D recordings.

Low-level red laser (LLRL)-tissue interactions have a wide range of medical applications and are garnering increased attention. Although the positive effects of low-level laser therapy (LLLT) have frequently been reported and enhanced collagen accumulation has been identified as one of the most important mechanisms involved, little is known about LLRL-collagen interactions. In this study, we aimed to investigate the influence of LLRL irradiation on collagen, in correlation with fibroblast response. Atomic force microscopy (AFM) and fluorescence spectroscopy were used to characterize surfaces and identify conformational changes in collagen before and after LLRL irradiation. Irradiated and non-irradiated collagen thin films were used as culturing substrates to investigate fibroblast response with fluorescence microscopy. The results demonstrated that LLRL induced small alterations in fluorescence emission and had a negligible effect on the topography of collagen thin films. However, fibroblasts cultured on LLRL-irradiated collagen thin films responded to LRLL. The results of this study show for the first time the effect of LLRL irradiation on pure collagen. Although irradiation did not affect the nanotopography of collagen, it influenced cell behavior. The role of collagen appears to be crucial in the LLLT mechanism, and our results demonstrated that LLRL directly affects collagen and indirectly affects cell behavior. PMID:26498450

Microstructure of undoped and phosphorus doped Si:H and Si,C:H films was analyzed by selected-area diffraction, conical dark-field imaging, energy-dispersive x-ray spectroscopy, and electron energy-loss spectroscopy in transmission electron microscopes. Thin films were synthesized by remote plasma-enhanced chemical vapor deposition and characterized in terms of degree of crystallinity. Distribution of phosphorus in Si:H and Si,C:H films, and of carbon in Si,C:H films was evaluated. Results indicate that (i) the microstructure of a film may be two phase, consisting of silicon microcrystallites in an amorphous matrix, (ii) phosphorus doping as well as the presence of carbon influences the degree of crystallinity by reducing the average size and volume fraction of microcrystallites, (iii) the presence of carbon and phosphorus doping completely suppresses the crystalline phase, (iv) phosphorus is distributed at approximately the same concentration in both the crystalline and amorphous phases of diphasic films, and (v) carbon is detected in the amorphous phase of the Si,C:H films.

Purpose: The texture analysis method is useful to estimate structural features of images as color, size, and shape. The study aims to determine a dose-response curve by texture analysis of Gafchromic EBT3 film images using scanning electron microscopy (SEM). Methods: The uncoated Gafchromic EBT3 films were prepared to directly scan over the active surface layer of EBT3 film using SEM. The EBT3 films were exposed at a dose range of 0 to 10 Gy using a 6 MV photon beam. The exposed film samples were SEM-scanned at 100X, 1000X, and 3000X magnifications. The four texture features (Homogeneity, Correlation, Contrast, and Energy) were calculated based on the gray level co-occurrence matrix (GLCM) derived from the SEM images at each dose. To validate a correlation between delivered doses and texture features, an R-squared value in linear regression was tested. Results: The results showed that the Correlation index was more suitable as dose indices than the other three texture features due to higher linearity and sensitivity of the dose response curves. Further the Correlation index of 3000X magnified SEM images with 9 pixel offsets had an R-squared value of 0.964. The differences between the delivered doses and the doses measured by this method were 0.9, 1.2, 0.2, and 0.2 Gy at 5, 10, 15, and 20 Gy, respectively. Conclusion: It seems to be feasible to convert micro-scale structural features of {sub χ}t{sub χχχ}he EBT3 films to absorbed doses using the texture analysis method.

Epitaxial CdTe thin films were grown on GaAs/Si(001) substrates by metalorganic chemical vapor deposition using thin GaAs as a buffer layer. The interfaces were investigated using high-resolution transmission electron microscopy and geometric phase analysis strain mapping. It was observed that dislocation cores exist at the CdTe/GaAs interface with periodic distribution. The spacing of the misfit dislocation was measured to be about 2 nm, corresponding to the calculated spacing of a misfit dislocation (2.6 nm) in CdTe/Si with Burgers vector of a[110]/2. From these results, it is suggested that the GaAs buffer layer effectively absorbs the strain originating from the large lattice mismatch between the CdTe thin film and Si substrate with the formation of periodic structural defects.

This work aims to simplify multi-dimensional femtosecond transient absorption microscopy (TAM) data into decay associated amplitude maps (DAAMs) that describe the spatial distributions of dynamical processes occurring on various characteristic timescales. Application of this method to TAM data obtained from a model methyl-ammonium lead iodide (CH3NH3PbI3) perovskite thin film allows us to simplify the data set comprising 68 time-resolved images into four DAAMs. These maps offer a simple means to visualize the complex electronic excited-state dynamics in this system by separating distinct dynamical processes evolving on characteristic timescales into individual spatial images. This approach provides new insight into subtle aspects of ultrafast relaxation dynamics associated with excitons and charge carriers in the perovskite thin film, which have recently been found to coexist at spatially distinct locations. PMID:27308671

This work aims to simplify multi-dimensional femtosecond transient absorption microscopy (TAM) data into decay associated amplitude maps (DAAMs) that describe the spatial distributions of dynamical processes occurring on various characteristic timescales. Application of this method to TAM data obtained from a model methyl-ammonium lead iodide (CH3NH3PbI3) perovskite thin film allows us to simplify the data set comprising 68 time-resolved images into four DAAMs. These maps offer a simple means to visualize the complex electronic excited-state dynamics in this system by separating distinct dynamical processes evolving on characteristic timescales into individual spatial images. This approach provides new insight into subtle aspects of ultrafast relaxation dynamics associated with excitons and charge carriers in the perovskite thin film, which have recently been found to coexist at spatially distinct locations.

Here, we present a direct observation of fatigue phenomena in epitaxially grown Pb(Zr{sub 0.2}Ti{sub 0.8})O{sub 3} (PZT) thin films using second harmonic piezoresponse force microscopy (SH-PFM). We observed strong correlation between the SH-PFM amplitude and phase signals with the remnant piezoresponse at different switching cycles. The SH-PFM results indicate that the average fraction of switchable domains decreases globally and the phase delays of polarization switching differ locally. In addition, we found that the fatigue developed uniformly over the whole area without developing region-by-region suppression of switchable polarization as in polycrystalline PZT thin films.

When developing a new formulation, the development, calibration and validation steps of analytical methods based on vibrational spectroscopy are time-consuming. For each new formulation, real samples must be produced and a "reference method" must be used in order to determine the Active Pharmaceutical Ingredient (API) content of each sample. To circumvent this issue, the paper presents a simple approach based on the film-casting technique used as a calibration tool in the framework of hot-melt extrusion process. Confocal Raman microscopic method was successfully validated for the determination of itraconazole content in film-casting samples. Then, hot-melt extrusion was carried out to produce real samples in order to confront the results obtained with confocal Raman microscopy and Ultra High Performance Liquid Chromatography (UHPLC). The agreement between both methods was demonstrated using a comparison study based on the Bland and Altman's plot. PMID:27154691

This work aims to simplify multi-dimensional femtosecond transient absorption microscopy (TAM) data into decay associated amplitude maps that describe the spatial distributions of dynamical processes occurring on various characteristic timescales. Application of this method to TAM data obtained from a model methyl-ammonium lead iodide (CH3NH3PbI3) perovskite thin film allows us to simplify the dataset consisting of a 68 time-resolved images into 4 decay associated amplitude maps. These maps provide a simple means to visualize the complex electronic excited-state dynamics in this system by separating distinct dynamical processes evolving on characteristic timescales into individual spatial images. This approach provides new insightmore » into subtle aspects of ultrafast relaxation dynamics associated with excitons and charge carriers in the perovskite thin film, which have recently been found to coexist at spatially distinct locations.« less

This paper presents in situ characterization of lithium-ion diffusion at nano- to micro-meter scales in a Li-rich layered oxide thin film cathode under external bias by using Electrochemical Strain Microscopy (ESM) and Atomic Force Microscopy (AFM) techniques. The local variations of the diffusion coefficient are calculated and visualized from the ESM images. The results indicate that the Li-ion movement is closely correlated with the changes in the surface topography when the Li-rich cathode is subjected to an external bias. Furthermore, bias-induced Li-ion redistribution is partially reversible. Topography evolution due to Li-ion diffusion and relaxation behaviour are observed. The results from this in situ study provide the insight into the Li-ion diffusion mechanism in the cathode material and pave the way for studying the details of the diffusion-related phenomenon in Li-ion battery materials. PMID:26242479

The peptide PcL342-354C was obtained from the Cry1Ab16 toxin present in Bacillus thuringiensis ("Computational Modeling Deduced Three Dimensional Structure of Cry1Ab16 Toxin from B. thuringiensis AC11" (Kashyap, 2012) [1]). In this data article, we report the synthesis and characterization of the PcL342-354C peptide by MALDI-TOF/TOF mass spectrometry. In addition, the preparation of layer-by-layer films is shown based on interspersion of this peptide with both polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate) (PSS), self-assembled on ITO (indium tin oxide) electrodes. The morphology of the ITO/PEI/PSS/PcL342-354C film was analyzed using atomic force microscopy (AFM). We also evaluated the effect of the number of bilayers in ITO/PEI/(PSS/PcL342-354C) n on the morphology of the film using AFM amplitude images. Further details about this study were published elsewhere, "Layer-by-layer films containing peptides of the Cry1Ab16 toxin from B. thuringiensis for potential biotechnological applications," (Plácido et al., 2016) [2]. PMID:27294178

Films of molybdenum grown on the (112¯0) plane of sapphire (Al 2O 3) are characterized using low-energy microscopy and low-energy electron diffraction. Stress fields observed on the Mo surface originate at dislocations and at miscut steps of the buried molybdenum-alumina vicinal interface. As-grown films contain small-angle grain boundaries. These are largely eliminated upon heating to 1700 K as edge dislocations that form the boundaries become extremely mobile. Edge dislocations attract and annihilate one another, and the small-angle grain boundaries disappear. Mobility of edge dislocations is correlated with rapid diffusion of carbon, which apparently pins dislocations up to temperatures that allow diffusion of carbon from dislocations into the bulk. The main contaminants of the Mo surface are carbon, oxygen and carbon monoxide. The most stable impurities are carbides that persist to 1700 K. Oxygen promotes bunching of monatomic steps into groups of two, three and four. Electron beams dissociate CO with energy less than 1 eV and deposit residues of carbon. Fairly ideal single crystal films of Mo produced by annealing exhibit monatomic surface step and terrace structure, and a minimum of dislocations. The quality of surfaces on these films exceeds that of typical single crystal bulk samples and is well suited for fundamental studies in surface science.

Materials for very high density magnetic storage media, with capacities of 10 Gbits/in{sup 2} and beyond, require high coercivity and high signal to noise ratio. To achieve storage densities of this level engineering of the material to produce very fine, magnetically decoupled domains is necessary. We have characterized the microstructure and microchemistry of 10 nm thick CoPt and CoPt + ZrO{sub x} thin films, as deposited and annealed, using transmission electron microscopy (TEM) and nanometer-scale energy dispersive x-ray spectroscopy (EDS).

Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

In this paper we describe the development and dissemination of a film-based educational intervention to reduce negative attitudes towards gay and bisexual men and transgender women (GBT) and people living with HIV/AIDS (PLWHA) in Latino communities, with a focus on youth. The intervention, Tal Como Somos/Just as We Are, is based on stigma and attribution theories, extensive formative research, and community input. Evaluation findings among educators and school youth suggest the film has the potential to effectively impact attitudes towards GBT and PLWHA. The film and intervention are being disseminated using diffusion of innovations theory through community-based organizations, schools, television broadcasting and film festivals. PMID:24377496

We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent.

We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent.

The report contains a detailed description of the experimental complexities encountered in developing scanning tunneling microscope (STM) probing of atomic structure on the surface of freshly-grown hydrogenated-amorphous semiconductors. It also contains a speculative microscopic film-growth model that explains differences between the disorder in CVD grown a-Ge:H versus a-Si:H films. This model is derived from prior results obtained in the chemical analysis of GeH{sub 4} plasmas, combined with surface reaction and thermodynamic considerations. The neutral radical fragments of silane, disilane and germane dissociation in discharges, which dominate the vapor and film-growth reactions, have been deduced from detailed analysis of prior data and are reported. 4 refs., 7 figs.